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1.
Chem Commun (Camb) ; 57(93): 12476-12479, 2021 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-34734602

RESUMO

We identified small-molecule enhancers of cellular stress granules by observing molecular crowding of proteins and RNAs in a time-dependent manner. Hit molecules sensitized the IRF3-mediated antiviral mechanism in the presence of poly(I:C) and inhibited the replication of SARS-CoV-2 by inducing stress granule formation. Thus, modulating multimolecular crowding can be a promising strategy against SARS-CoV-2.


Assuntos
Antivirais/farmacologia , Benzopiranos/farmacologia , Grânulos Citoplasmáticos/efeitos dos fármacos , Pirazóis/farmacologia , SARS-CoV-2/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/química , Benzopiranos/química , Linhagem Celular Tumoral , Chlorocebus aethiops , Grânulos Citoplasmáticos/metabolismo , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Humanos , Fator Regulador 3 de Interferon/metabolismo , Lopinavir/farmacologia , Testes de Sensibilidade Microbiana , Estrutura Molecular , Poli I-C/farmacologia , Pirazóis/química , Relação Estrutura-Atividade , Células Vero
2.
J Immunol ; 207(9): 2310-2324, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34551966

RESUMO

IFN-γ, a proinflammatory cytokine produced primarily by T cells and NK cells, activates macrophages and engages mechanisms to control pathogens. Although there is evidence of IFN-γ production by murine macrophages, IFN-γ production by normal human macrophages and their subsets remains unknown. Herein, we show that human M1 macrophages generated by IFN-γ and IL-12- and IL-18-stimulated monocyte-derived macrophages (M0) produce significant levels of IFN-γ. Further stimulation of IL-12/IL-18-primed macrophages or M1 macrophages with agonists for TLR-2, TLR-3, or TLR-4 significantly enhanced IFN-γ production in contrast to the similarly stimulated M0, M2a, M2b, and M2c macrophages. Similarly, M1 macrophages generated from COVID-19-infected patients' macrophages produced IFN-γ that was enhanced following LPS stimulation. The inhibition of M1 differentiation by Jak inhibitors reversed LPS-induced IFN-γ production, suggesting that differentiation with IFN-γ plays a key role in IFN-γ induction. We subsequently investigated the signaling pathway(s) responsible for TLR-4-induced IFN-γ production in M1 macrophages. Our results show that TLR-4-induced IFN-γ production is regulated by the ribosomal protein S6 kinase (p70S6K) through the activation of PI3K, the mammalian target of rapamycin complex 1/2 (mTORC1/2), and the JNK MAPK pathways. These results suggest that M1-derived IFN-γ may play a key role in inflammation that may be augmented following bacterial/viral infections. Moreover, blocking the mTORC1/2, PI3K, and JNK MAPKs in macrophages may be of potential translational significance in preventing macrophage-mediated inflammatory diseases.


Assuntos
Interferon gama/biossíntese , Macrófagos/efeitos dos fármacos , Poli I-C/farmacologia , COVID-19/imunologia , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/imunologia , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , MAP Quinase Quinase Quinases/antagonistas & inibidores , MAP Quinase Quinase Quinases/imunologia , Macrófagos/imunologia , Fosfatidilinositol 3-Quinases/imunologia , Proteínas Quinases S6 Ribossômicas 70-kDa/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas 70-kDa/imunologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/imunologia , Receptor 4 Toll-Like/agonistas
3.
Biomed Khim ; 67(4): 331-337, 2021 Jul.
Artigo em Russo | MEDLINE | ID: mdl-34414891

RESUMO

The pathogenetic mechanisms associated with alcohol use include dysregulation of the innate immune system mechanisms in the brain. TLR3 expression is increased in the postmortem material of the prefrontal cortex of humans. An increase in the TLR3 signaling activity leads to the induction of interferons (IFN). IFN are associated with depressive symptoms and, therefore, may play a role in the pathogenesis of alcoholism; however, the exact mechanisms of intracellular signaling mediated by the influence of ethanol are not fully elucidated and their study was the purpose of this work. The experimental results showed that ethanol and the TLR3 agonist Poly (I:C) increased the content of TLR3, IFNß, and IFNγ mRNA in the prefrontal cortex. In addition, expression of the TRAIL encoding gene also increased, and this increase positively correlaed with the mRNA content of TLR3, IFNß and IFNγ both under alcoholization conditions and after injections of the TLR3 agonist. The data obtained may indicate that alcoholization is able to activate TLR3-TRAIL-IFN-signaling in the prefrontal cortex of the brain.


Assuntos
Interferons , Receptor 3 Toll-Like , Animais , Encéfalo/metabolismo , Poli I-C/farmacologia , Ratos , Transdução de Sinais , Receptor 3 Toll-Like/genética , Receptor 3 Toll-Like/metabolismo
4.
Biomolecules ; 11(8)2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34439814

RESUMO

Vitamin C is well documented to have antiviral functions; however, there is limited information about its effect on airway epithelial cells-the first cells to encounter infections. Here, we examined the effect of vitamin C on human bronchial epithelium transformed with Ad12-SV40 2B (BEAS-2B) cells, and observed that sodium-dependent vitamin C transporter 2 (SVCT2) was the primary vitamin C transporter. Transcriptomic analysis revealed that treating BEAS-2B cells with vitamin C led to a significant upregulation of several metabolic pathways and interferon-stimulated genes (ISGs) along with a downregulation of pathways involved in lung injury and inflammation. Remarkably, vitamin C also enhanced the expression of the viral-sensing receptors retinoic acid-inducible gene 1 (RIG-1) and melanoma differentiation-associated protein 5 (MDA-5), which was confirmed at the protein and functional levels. In addition, the lungs of l-gulono-γ-lactone oxidase knockout (GULO-KO) mice also displayed a marked decrease in these genes compared to wild-type controls. Collectively, our findings indicate that vitamin C acts at multiple levels to exert its antiviral and protective functions in the lungs.


Assuntos
Antivirais/farmacologia , Ácido Ascórbico/farmacologia , Células Epiteliais/efeitos dos fármacos , Helicase IFIH1 Induzida por Interferon/genética , Receptores do Ácido Retinoico/genética , Transportadores de Sódio Acoplados à Vitamina C/genética , Animais , Transporte Biológico , Brônquios/efeitos dos fármacos , Brônquios/metabolismo , Linhagem Celular Transformada , Proteína DEAD-box 58/genética , Proteína DEAD-box 58/metabolismo , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Humanos , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Helicase IFIH1 Induzida por Interferon/metabolismo , Interferon-alfa/antagonistas & inibidores , Interferon-alfa/farmacologia , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , L-Gulonolactona Oxidase/deficiência , L-Gulonolactona Oxidase/genética , Camundongos , Camundongos Knockout , Proteínas de Resistência a Myxovirus/genética , Proteínas de Resistência a Myxovirus/metabolismo , Poli I-C/antagonistas & inibidores , Poli I-C/farmacologia , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Receptores do Ácido Retinoico/metabolismo , Transportadores de Sódio Acoplados à Vitamina C/metabolismo , Transcriptoma
5.
Cells ; 10(8)2021 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-34440683

RESUMO

Apolipoprotein L1 (APOL1) high-risk genotypes (HRG), G1 and G2, increase the risk of various non-diabetic kidney diseases in the African population. To date, the precise mechanisms by which APOL1 risk variants induce injury on podocytes and other kidney cells remain unclear. Trying to unravel these mechanisms, most studies have used animal or cell models created by gene editing. We developed and characterised conditionally immortalised human podocyte cell lines derived from urine of a donor carrying APOL1 HRG G2/G2. Following induction of APOL1 expression by polyinosinic-polycytidylic acid (poly(I:C)), we assessed functional features of APOL1-induced podocyte dysfunction. As control, APOL1 wild type (G0/G0) podocyte cell line previously generated from a Caucasian donor was used. Upon exposure to poly(I:C), G2/G2 and G0/G0 podocytes upregulated APOL1 expression resulting in podocytes detachment, decreased cells viability and increased apoptosis rate in a genotype-independent manner. Nevertheless, G2/G2 podocyte cell lines exhibited altered features, including upregulation of CD2AP, alteration of cytoskeleton, reduction of autophagic flux and increased permeability in an in vitro model under continuous perfusion. The human APOL1 G2/G2 podocyte cell model is a useful tool for unravelling the mechanisms of APOL1-induced podocyte injury and the cellular functions of APOL1.


Assuntos
Apolipoproteína L1/metabolismo , Modelos Biológicos , Adulto , Apolipoproteína L1/genética , Autofagia/efeitos dos fármacos , Adesão Celular , Linhagem Celular , Pré-Escolar , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Feminino , Genótipo , Humanos , Nefropatias/metabolismo , Nefropatias/patologia , Masculino , Podócitos/citologia , Podócitos/metabolismo , Poli I-C/farmacologia , Regulação para Cima/efeitos dos fármacos
6.
Cells ; 10(8)2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34440679

RESUMO

The liver with resident tissue macrophages is the site of vivid innate immunity, activated also by pathogen-associated molecular patterns (PAMPs) leaking through the intestinal barrier. As gut-derived inflammatory diseases are of outstanding importance in broiler chickens, the present study aimed to establish a proper hepatic inflammatory model by comparing the action of different PAMPs from poultry pathogens on chicken 2D and 3D primary hepatocyte-non-parenchymal cell co-cultures, the latter newly developed with a magnetic bioprinting method. The cultures were challenged by the bacterial endotoxins lipopolysaccharide (LPS) from Escherichia coli, lipoteichoic acid (LTA) from Staphylococcus aureus and by enterotoxin (ETxB) from Escherichia coli, Salmonella Typhimurium derived flagellin, phorbol myristate acetate (PMA) as a model proinflammatory agent and polyinosinic polycytidylic acid (poly I:C) for mimicking viral RNA exposure. Cellular metabolic activity was assessed with the CCK-8 test, membrane damage was monitored with the lactate dehydrogenase (LDH) leakage assay and interleukin-6 and -8 (Il-6 and -8) concentrations were measured in cell culture medium with a chicken specific ELISA. Both LPS and LTA increased the metabolic activity of the 3D cultures, concomitantly decreasing the LDH leakage, while in 2D cultures ETxB stimulated, PMA and poly I:C depressed the metabolic activity. Based on the moderately increased extracellular LDH activity, LTA seemed to diminish cell membrane integrity in 2D and poly I:C in both cell culture models. The applied endotoxins remarkably reduced the IL-8 release of 3D cultured cells, suggesting the effective metabolic adaptation and the presumably initiated anti-inflammatory mechanisms of the 3D spheroids. Notwithstanding that the IL-6 and IL-8 production of 2D cells was mostly not influenced by the endotoxins used, only the higher LTA dose was capable to evoke an IL-8 surge. Flagellin, PMA and poly I:C exerted proinflammatory action in certain concentrations in both 2D and 3D cultures, reflected by the increased cellular IL-6 release. Based on these data, LTA, flagellin, PMA and poly I:C can be considered as potent candidates to induce inflammation in chicken primary hepatic cell cultures, while LPS failed to trigger proinflammatory cytokine production, suggesting the relatively high tolerance of avian liver cells to certain bacterial endotoxins. These results substantiate that the established 3D co-cultures seemed to be proper tools for testing potential proinflammatory molecules; however, the remarkable differences between 2D and 3D models should be addressed and further studied.


Assuntos
Galinhas/imunologia , Imunidade Inata/efeitos dos fármacos , Fígado/efeitos dos fármacos , Padrões Moleculares Associados a Patógenos/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Galinhas/metabolismo , Técnicas de Cocultura , Enterotoxinas/farmacologia , Flagelina/farmacologia , Mediadores da Inflamação/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Lipopolissacarídeos/farmacologia , Fígado/imunologia , Fígado/metabolismo , Masculino , Poli I-C/farmacologia , Esferoides Celulares , Ácidos Teicoicos/farmacologia , Acetato de Tetradecanoilforbol/farmacologia
7.
Fish Shellfish Immunol ; 116: 150-160, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34265416

RESUMO

As a tyrosine phosphatase, Src homology 2-containing protein tyrosine phosphatase 2 (SHP2) serves as an inhibitor in PI3K-Akt pathway. In mammals, SHP2 can phosphorylate GSK3ß at Y216 site to control the expression of IFN. So far, the multiple functions of SHP2 have been reported in mammals. However, little is known about fish SHP2. In this study, we cloned and identified a grass carp (Ctenopharyngodon idellus) SHP2 gene (CiSHP2, MT373151). SHP2 is conserved among different vertebrates by amino acid sequences alignment and the phylogenetic tree analysis. CiSHP2 shared the closest homology with Danio rerio SHP2. Simultaneously, SHP2 was also tested in grass carp tissues and CIK (C. idellus kidney) cells. We found that it responded to poly I:C stimulation. CiSHP2 was located in the cytoplasm just as the same as those of mammals. Interestingly, it inhibited the phosphorylation level of GSK3ß in a non-contact manner. Meanwhile CiGSK3ß interacted with and directly phosphorylated CiTBK1. In addition, we found that CiSHP2 also reduced the phosphorylation level of CiTBK1 by CiGSK3ß, and then it depressed the expression of IFN I via GSK3ß-TBK1 axis. These results suggested that CiSHP2 was involved in CiGSK3ß and CiTBK1 activity but not regulated their transcriptional level. At the same time, we also found that CiSHP2 also influenced the activity of CiIRF3. Therefore, fish SHP2 inhibited IFN I expression through blocking GSK3ß-TBK1 signal axis.


Assuntos
Carpas/imunologia , Proteínas de Peixes/imunologia , Glicogênio Sintase Quinase 3 beta/imunologia , Interferon Tipo I/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/imunologia , Sequência de Aminoácidos , Animais , Carpas/genética , Linhagem Celular , Proteínas de Peixes/genética , Fosforilação , Filogenia , Poli I-C/farmacologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética
8.
Front Immunol ; 12: 626895, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34267744

RESUMO

In mammals, Interleukin-17 cytokine family plays critical roles in both acute and chronic inflammatory responses. In fish species, three Interleukin-17A/F (IL-17A/F) genes have been identified to be homologous to mammalian IL-17A and IL-17F, but little is known about their functional activity. In this study, Pf_IL-17A/F1, 2 and 3 genes were cloned from yellow catfish (Pelteobagrus fulvidraco) and they differed in protein structure and exon length, implying that they may have divergent bioactivity. Real-time quantitative PCR analyses revealed that three Pf_IL-17A/F genes were highly expressed in blood and mucosal tissues (skin+mucus and gill) from healthy adult fish. The mRNA expressions of Pf_IL-17A/F1, 2 and 3 genes were significantly up-regulated in the gill, skin+mucus, head kidney and spleen after challenge with Edwardsiella ictaluri and in the isolated peripheral blood leucocytes (PBLs) of yellow catfish after stimulation with phytohaemagglutinin (PHA), lipopolysaccharides (LPS), peptidoglycan (PGN) and polyinosinic-polycytidylic acid (Poly I:C). These results indicate that Pf_IL-17A/F1, 2 and 3 genes may play a vital role in the regulation of immune against pathogens. Additionally, the recombinant (r) Pf_IL-17A/F1, 2 and 3 proteins significantly induced the mRNA expressions of proinflammatory cytokines, chemokines and antibacterial peptides genes, and the rPf_IL-17A/F 2 and 3 proteins promoted phagocytosis of PBLs more powerfully than the rPf_IL-17A/F1. Furthermore, the rPf_IL-17A/F1, 2 and 3 proteins might activate the NF-κB and MAPK signal pathways by IL-17RA, ACT1, TRAF6, TRAF2, TRAF5 and TAK1, indicating that the three Pf_IL-17A/F proteins may play different roles in promoting inflammatory response.


Assuntos
Peixes-Gato/genética , Peixes-Gato/imunologia , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Interleucina-17/genética , Interleucina-17/imunologia , Animais , Rim Cefálico/imunologia , Interleucina-17/química , Interleucina-17/classificação , Leucócitos/efeitos dos fármacos , Leucócitos/imunologia , Lipopolissacarídeos/farmacologia , Peptidoglicano/farmacologia , Fito-Hemaglutininas/farmacologia , Poli I-C/farmacologia , Baço/imunologia
9.
Fish Shellfish Immunol ; 115: 75-85, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34091036

RESUMO

Thioredoxin domain-containing protein 17 (TXNDC17) is an important, highly conserved oxidoreductase protein, ubiquitously expressed in all living organisms. It is a small (~14 kDa) protein mostly co-expressed with thioredoxin 1 (TRx1). In the present study, we obtained the TXNDC17 gene sequence from a previously constructed yellowtail clownfish (Amphiprion clarkii) (AcTXNDC17) database and studied its phylogeny as well as the protein's molecular characteristics, antioxidant, and antiapoptotic effects. The full length of the AcTXNDC17 cDNA sequence was 862 bp with a 372 bp region encoding a 123 amino acid (aa) protein. The predicted molecular mass and isoelectric point of AcTXNDC17 were 14.2 kDa and 5.75, respectively. AcTXNDC17 contained a TRX-related protein 14 domain and a highly conserved N-terminal Cys43-Pro44-Asp45-Cys46 motif. qPCR analysis revealed that AcTXNDC17 transcripts were ubiquitously and differently expressed in all the examined tissues. AcTXNDC17 expression in the spleen tissue was significantly upregulated in a time-dependent manner upon stimulation with lipopolysaccharide (LPS), polyinosinic-polycytidylic (poly I:C), and Vibrio harveyi. Besides, LPS-induced intrinsic apoptotic pathway (TNF-α, caspase-8, Bid, cytochrome C, caspase-9, and caspase-3) gene expression was significantly lower in AcTXNDC17-overexpressing RAW264.7 cells, as were NF-κB activation and nitric oxide (NO) production. Furthermore, the viability of H2O2-stimulated macrophages was significantly improved under AcTXNDC17 overexpression. Collectively, our findings indicate that AcTXNDC17 is involved in the innate immune response of the yellowtail clownfish.


Assuntos
Doenças dos Peixes/imunologia , Peixes/genética , Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Tiorredoxinas/genética , Tiorredoxinas/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Lipopolissacarídeos/farmacologia , Filogenia , Poli I-C/farmacologia , Alinhamento de Sequência/veterinária , Tiorredoxinas/química , Vibrio/fisiologia , Vibrioses/imunologia
10.
Poult Sci ; 100(8): 101247, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34174563

RESUMO

Exosomes are small membrane vesicles that contain proteins and nucleic acids derived from secretory cells and mediate intracellular communication. Immune cell-derived exosomes regulate immune responses and gene expression of recipient cells. Macrophages recognize viral dsRNA via Toll-like receptor 3, thereby inducing the activation of transcription factors such as interferon regulatory factor 3 and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). In this study, we aimed to identify the immunomodulatory functions of exosomes derived from chicken macrophages (HD11) stimulated with polyinosinic-polycytidylic acid (poly[I:C]); exosomes were then delivered into HD11 cells and CU91 chicken T cells. Exosomes purified from poly(I:C)-activated macrophages stimulated the expression of type I interferons, proinflammatory cytokines, anti-inflammatory cytokines, and chemokines in HD11 and CU91 cells. Moreover, poly(I:C)-stimulated exosomes induced the NF-κB signaling pathway by phosphorylating TAK1 and NF-κB1. Therefore, we suggest that after the activation of Toll-like receptor 3 ligands following infection with dsRNA virus, chicken macrophages regulate the immune response of naive macrophages and T cells through the NF-κB signaling pathway. Furthermore, poly(I:C)-activated exosomes can be potentially utilized as immunostimulators.


Assuntos
Exossomos , Poli I-C , Animais , Galinhas , Imunidade , Macrófagos , NF-kappa B , Poli I-C/farmacologia
11.
Nat Commun ; 12(1): 3958, 2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-34172753

RESUMO

Astrocytes play important roles in neurological disorders such as stroke, injury, and neurodegeneration. Most knowledge on astrocyte biology is based on studies of mouse models and the similarities and differences between human and mouse astrocytes are insufficiently characterized, presenting a barrier in translational research. Based on analyses of acutely purified astrocytes, serum-free cultures of primary astrocytes, and xenografted chimeric mice, we find extensive conservation in astrocytic gene expression between human and mouse samples. However, the genes involved in defense response and metabolism show species-specific differences. Human astrocytes exhibit greater susceptibility to oxidative stress than mouse astrocytes, due to differences in mitochondrial physiology and detoxification pathways. In addition, we find that mouse but not human astrocytes activate a molecular program for neural repair under hypoxia, whereas human but not mouse astrocytes activate the antigen presentation pathway under inflammatory conditions. Here, we show species-dependent properties of astrocytes, which can be informative for improving translation from mouse models to humans.


Assuntos
Astrócitos/fisiologia , Animais , Apresentação do Antígeno , Astrócitos/efeitos dos fármacos , Células Cultivadas , Expressão Gênica/efeitos dos fármacos , Humanos , Inativação Metabólica , Inflamação , Camundongos , Mitocôndrias/metabolismo , Doenças do Sistema Nervoso/tratamento farmacológico , Doenças do Sistema Nervoso/patologia , Estresse Oxidativo , Poli I-C/farmacologia , Poli I-C/uso terapêutico , Especificidade da Espécie , Transcriptoma/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Fator de Necrose Tumoral alfa/uso terapêutico
12.
Int J Biol Macromol ; 185: 176-193, 2021 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-34144067

RESUMO

Inflammation is a common manifestation of body immunity and mediates a cascade of cytokines. Tumor necrosis factor-α (TNF-α), as a multi-effect cytokine, plays an important role in the inflammatory response by interacting with its receptor (TNFR). In this study, Pf_TNF-α, Pf_TNFR1 and Pf_TNFR2 genes were cloned from yellow catfish (Pelteobagrus fulvidraco), and bioinformatics analyses showed that the three genes were conserved and possessed similar sequence characteristics as those of other vertebrates. The qPCR results showed that Pf_TNF-α, Pf_TNFR1 and Pf_TNFR2 mRNAs were constitutively expressed in 14 tissues and the lymphocytes of four tissues from healthy adults. The mRNA expression levels of Pf_TNF-α and Pf_TNFR1 genes were significantly up-regulated in the spleen, liver, trunk kidney, head kidney and gill after Edwardsiella ictaluri infection, while the mRNA expression of Pf_TNFR2 was significantly up-regulated in the spleen, and down-regulated in the liver and gill. In the isolated peripheral blood leukocytes (PBLs) of yellow catfish, the expression of Pf_TNF-α mRNA was notably up-regulated and the two Pf_TNFR transcripts were distinctly down-regulated after stimulation with lipopolysaccharides (LPS), peptidoglycan (PGN), polyinosinic-polycytidylic acid (Poly I:C) and phytohaemagglutinin (PHA). After stimulated by recombinant (r) Pf_sTNF protein, the mRNA expressions of various inflammatory factors genes were up-regulated in the PBLs. Meanwhile, rPf_sTNF promoted the phagocytic activity of leukocytes, whereas the activity mediated by rPf_sTNF could be inhibited by rPf_TNFR1CRD2/3 and rPf_TNFR2CRD2/3. The up-regulation of TNF-α and IL-1ß mRNAs expression triggered by rPf_sTNF could be inhibited by MAPK inhibitor (VX-702) and NF-κB inhibitor (PDTC). rPf_sTNF induced the expression of FADD mRNA in PBLs and increased the apoptotic rate of PBLs, and inhibiting the NF-κB and MAPK signal pathways could enhance the apoptosis of PBLs. The results indicate that Pf_TNF-α, Pf_TNFR1 and Pf_TNFR2 play important roles in the immune response of yellow catfish to bacterial invasion.


Assuntos
Peixes-Gato/genética , Clonagem Molecular/efeitos dos fármacos , Receptores do Fator de Necrose Tumoral/genética , Fator de Necrose Tumoral alfa/genética , Animais , Biologia Computacional , Feminino , Proteínas de Peixes/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Masculino , Especificidade de Órgãos , Peptidoglicano/farmacologia , Filogenia , Fito-Hemaglutininas/farmacologia , Poli I-C/farmacologia
13.
Fish Shellfish Immunol ; 116: 61-73, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34157396

RESUMO

In aquaculture, commercial fish such as red hybrid tilapia are usually raised at high density to boost the production within a short period of time. This overcrowded environment, however, may cause stress to the cultured fish and increase susceptibility to infectious diseases. Antibiotics and chemotherapeutics are used by fish farmers to overcome these challenges, but this may increase the production cost. Studies have reported on the potential of mushroom polysaccharides that can act as immunostimulants to enhance the immune response and disease resistance in fish. In the current study, hot water extract (HWE) from mushroom stalk waste (MSW) was used to formulate fish feed and hence administered to red hybrid tilapia to observe the activation of immune system. Upon 30 days of feeding, the fish were challenged with pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharides (LPS) and polyinosinic:polycytidylic acid (poly (I:C)) to mimic bacterial and viral infection, respectively. HWE supplementation promoted better feed utilisation in red hybrid tilapia although it did not increase the body weight gain and specific growth rate compared to the control diet. The innate immunological parameters such as phagocytic activity and respiratory burst activity were significantly higher in HWE-supplemented group than that of the control group following PAMPs challenges. HWE-supplemented diet also resulted in higher mRNA transcription of il1b and tnfa in midgut, spleen and head kidney at 1-day post PAMPs injection. Tlr3 exhibited the highest upregulation in the HWE fed fish injected with poly (I:C). At 3-days post PAMPs injection, both ighm and tcrb expression were upregulated significantly in the spleen and head kidney. Results showed that HWE supplementation enhances the immune responses of red hybrid tilapia and induced a higher serum bactericidal activity against S. agalactiae.


Assuntos
Ciclídeos , Misturas Complexas/farmacologia , Suplementos Nutricionais , Lipopolissacarídeos/farmacologia , Padrões Moleculares Associados a Patógenos/farmacologia , Pleurotus , Poli I-C/farmacologia , Ração Animal , Animais , Quimera , Ciclídeos/genética , Ciclídeos/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Rim Cefálico/efeitos dos fármacos , Rim Cefálico/imunologia , Temperatura Alta , Imunidade Inata/efeitos dos fármacos , Interleucina-1beta/genética , Leucócitos/efeitos dos fármacos , Leucócitos/imunologia , Fagocitose/efeitos dos fármacos , Baço/efeitos dos fármacos , Baço/imunologia , Streptococcus agalactiae/imunologia , Fator de Necrose Tumoral alfa/genética , Resíduos , Água
14.
Mol Immunol ; 136: 110-117, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34098343

RESUMO

Reticuloendothelial virus (REV) is widely found in many domestic poultry areas and results in severe immunosuppression of infected chickens. This increases the susceptibility to other pathogens, which causes economic losses to the poultry industry. The aim of our study was to determine whether polyinosinic-polycytidylic acid [Poly (I: C)] treatment could inhibit REV replication in chicken macrophage-like cell line, HD11. We found that Poly (I: C) treatment could markedly inhibit REV replication in HD11 from 24 to 48 h post infection (hpi). Additionally, Poly (I: C) treatment could switch HD11 from an inactive type into M1-like polarization from 24 to 48 hpi. Furthermore, Poly (I: C) treatment promoted interferon-ß secretion from HD11 post REV infection. Moreover, Toll-like receptor-3 (TLR-3) mRNA and protein levels in HD11 treated with Poly (I: C) were markedly increased compared to those of HD11 not treated with Poly (I: C). The above results suggested that Poly (I: C) treatment switches HD11 into M1-like polarization to secret more interferon-ß and activate TLR-3 signaling, which contributes to block REV replication. Our findings provide a theoretical reference for further studying the underlying pathogenic mechanism of REV and Poly (I: C) as a potential therapeutic intervention against REV infection.


Assuntos
Antivirais/farmacologia , Indutores de Interferon/farmacologia , Poli I-C/farmacologia , Vírus da Reticuloendoteliose Aviária/crescimento & desenvolvimento , Receptor 3 Toll-Like/metabolismo , Replicação Viral/efeitos dos fármacos , Animais , Linhagem Celular , Galinhas , Interferon beta/biossíntese , Interferon beta/metabolismo , Macrófagos/imunologia , Macrófagos/virologia , Vírus da Reticuloendoteliose Aviária/efeitos dos fármacos , Infecções por Retroviridae/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Infecções Tumorais por Vírus/tratamento farmacológico
15.
Invest Ophthalmol Vis Sci ; 62(7): 21, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-34144609

RESUMO

Purpose: For this study we aimed to understand if retinal pigment epithelial (RPE) cells express antimicrobial peptide lysozyme as a mechanism to protect the neuroretina from blood-borne pathogens. Methods: The expression of lysozyme in human and mouse RPE cells was examined by RT-PCR or immune (cyto)histochemistry in cell cultures or retinal sections. RPE cultures were treated with different concentrations of Pam3CSK4, lipopolysaccharides (LPS), staphylococcus aureus-derived peptidoglycan (PGN-SA), Poly(I:C), and Poly(dA:dT). The mRNA expression of lysozyme was examined by qPCR and protein expression by ELISA. Poly(I:C) was injected into the subretinal space of C57BL/6J mice and eyes were collected 24 hours later and processed for the evaluation of lysozyme expression by confocal microscopy. Bactericidal activity was measured in ARPE19 cells following LYZ gene deletion using Crispr/Cas9 technology. Results: The mRNA and protein of lysozyme were detected in mouse and human RPE cells under normal conditions, although the expression levels were lower than mouse microglia BV2 or human monocytes THP-1 cells, respectively. Immunohistochemistry showed punctate lysozyme expression inside RPE cells. Lysozyme was detected by ELISA in normal RPE lysates, and in live bacteria-treated RPE supernatants. Treatment of RPE cells with Pam3CSK4, LPS, PGN-SA, and Poly(I:C) enhanced lysozyme expression. CRISPR/Cas9 deletion of lysozyme impaired bactericidal activity of ARPE19 cells and reduced their response to LPS and Poly(I:C) stimulation. Conclusions: RPE cells constitutively express antimicrobial peptide lysozyme and the expression is modulated by pathogenic challenges. RPE cells may protect the neuroretina from blood-borne pathogens by producing antimicrobial peptides, such as lysozyme.


Assuntos
Lipopeptídeos/fisiologia , Muramidase , Retina , Animais , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Barreira Hematorretiniana/imunologia , Barreira Hematorretiniana/metabolismo , Células Cultivadas , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Camundongos , Muramidase/genética , Muramidase/farmacologia , Poli I-C/metabolismo , Poli I-C/farmacologia , Fatores de Proteção , Retina/imunologia , Retina/metabolismo , Epitélio Pigmentado da Retina/fisiologia
16.
Biochem Biophys Res Commun ; 565: 64-71, 2021 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-34098313

RESUMO

Neutrophil extracellular traps (NETs) are extracellular webs of DNA, histones and granular contents that are released by neutrophils to control infections. However, NETs that is not properly regulated can propagate inflammation and thrombosis. It was recognized that viruses can induce NETs. As a synthetic analog of viral double-stranded (ds) RNA, polyinosinic-polycytidylic acid [poly(I:C)] is known to induce inflammation and thrombosis. However, whether and how poly(I:C) modulates NETs remains unclear. Here, we have demonstrated that poly(I:C) induced extracellular DNA traps in human neutrophils in a dose-dependent manner. Further, poly(I:C) or dsRNA virus elevated the levels of myeloperoxidase-DNA complexes and citrullinated histone H3, which are specific markers of NETs, in both neutrophil supernatants and mouse plasma. Interestingly, a potent peptidylarginine deiminase 4 (PAD4) inhibitor, BB-CL-Amidine (BB-CLA) or PAD4 knockdown effectively prevented poly(I:C)-induced NETs formation and release. In addition, BB-CLA abrogated poly(I:C)-triggered neutrophil activation and infiltration, and vascular permeability in lungs. BB-CLA also attenuated poly(I:C)-induced thrombocytopenia in circulation, fibrin deposition and thrombus formation in tissues. Taken together, these results suggest that viral mimetic poly(I:C) may induce NETs-dependent inflammation and thrombosis through PAD4, and that inhibiting PAD4 may become a good strategy to protect against viral infection-caused inflammation/thrombosis-related pathological conditions of diseases.


Assuntos
Armadilhas Extracelulares/efeitos dos fármacos , Inflamação/metabolismo , Neutrófilos/efeitos dos fármacos , Poli I-C/farmacologia , Proteína-Arginina Desiminase do Tipo 4/metabolismo , Trombose/metabolismo , Amidinas/farmacologia , Animais , Células Cultivadas , Chlorocebus aethiops , Humanos , Inflamação/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ativação de Neutrófilo/efeitos dos fármacos , Neutrófilos/metabolismo , Proteína-Arginina Desiminase do Tipo 4/antagonistas & inibidores , Trombose/patologia
17.
EMBO J ; 40(15): e107176, 2021 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-34124789

RESUMO

Dendritic cell (DC) activation by viral RNA sensors such as TLR3 and MDA-5 is critical for initiating antiviral immunity. Optimal DC activation is promoted by type I interferon (IFN) signaling which is believed to occur in either autocrine or paracrine fashion. Here, we show that neither autocrine nor paracrine type I IFN signaling can fully account for DC activation by poly(I:C) in vitro and in vivo. By controlling the density of type I IFN-producing cells in vivo, we establish that instead a quorum of type I IFN-producing cells is required for optimal DC activation and that this process proceeds at the level of an entire lymph node. This collective behavior, governed by type I IFN diffusion, is favored by the requirement for prolonged cytokine exposure to achieve DC activation. Furthermore, collective DC activation was found essential for the development of innate and adaptive immunity in lymph nodes. Our results establish how collective rather than cell-autonomous processes can govern the initiation of immune responses.


Assuntos
Células Dendríticas/fisiologia , Interferon Tipo I/metabolismo , Linfonodos/citologia , Percepção de Quorum/fisiologia , Animais , Linfócitos T CD8-Positivos/fisiologia , Contagem de Células , Células Dendríticas/efeitos dos fármacos , Imunidade Inata/imunologia , Inflamação/patologia , Fator Regulador 7 de Interferon/genética , Fator Regulador 7 de Interferon/imunologia , Interferon Tipo I/farmacologia , Linfonodos/imunologia , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Camundongos Transgênicos , Poli I-C/farmacologia
18.
Front Immunol ; 12: 689783, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34168656

RESUMO

Interferon (IFN) system is considered as the first defense line against viral infection, and it has been extensively studied in vertebrates from fish to mammals. In invertebrates, Vagos from arthropod and IFN-like protein (CgIFNLP) from Crassostrea gigas appeared to function as IFN-like antiviral cytokines. In the present study, the CgIFNLP protein in hemocytes was observed to increase after Poly (I:C) stimulation. After CgIFNLP was knocked down by RNAi, the mRNA expression of IFN-stimulated genes (CgISGs) was significantly inhibited. Both cyclic GMP-AMP synthase (CgcGAS) and stimulator of interferon gene (CgSTING) identified from oyster were able to recognize the double-stranded nucleic acid [Poly (I:C) and dsDNA] and expressed at high level after Poly (I:C) stimulation. The expression of CgIFNLP and interferon regulatory factors (CgIRF1/8) and the nuclear translocation of CgIRF8 were all suppressed in CgcGAS-RNAi or CgSTING-RNAi oysters after Poly (I:C) stimulation. The expression level of CgSTING and TANK binding kinase1 (CgTBK1) did not decrease in CgcGAS-RNAi oysters. After CgSTING was knocked down, the high expression of CgTBK1 induced by Poly (I:C) was prevented significantly. These results indicated that there was a primitive IFN-like antiviral mechanism dependent on the cGAS/STING-TBK1-IRFs regulatory axis in mollusks, which was different from the classic cGAS-STING-TBK1 signal pathway in mammals.


Assuntos
Crassostrea/enzimologia , Imunidade , Fatores Reguladores de Interferon/metabolismo , Proteínas de Membrana/metabolismo , Nucleotidiltransferases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Crassostrea/efeitos dos fármacos , Crassostrea/imunologia , Crassostrea/virologia , Vírus de DNA/imunologia , Interações Hospedeiro-Patógeno , Imunidade/efeitos dos fármacos , Fatores Reguladores de Interferon/genética , Proteínas de Membrana/genética , Nucleotidiltransferases/genética , Poli I-C/farmacologia , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais
19.
Sci Rep ; 11(1): 10447, 2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-34001933

RESUMO

Microglia, CNS resident innate immune cells, respond strongly to activation of TLR3 and TLR4, which recognize viral dsRNA poly(I:C) and bacterial endotoxin LPS, respectively. However, few studies have thoroughly and parallelly compared functional phenotypes and downstream mechanisms between LPS- and poly(I:C)-exposed primary microglia. Here, we investigated the responses of mouse primary microglia upon LPS and poly(I:C) stimulation by detecting various phenotypes ranging from morphology, proliferation, secretion, chemotaxis, to phagocytosis. Furthermore, we explored their sequential gene expression and the downstream signal cascades. Interestingly, we found that the microglial activation pattern induced by LPS was distinguished from that induced by poly(I:C). Regarding microglial morphology, LPS caused an ameboid-like shape while poly(I:C) induced a bushy shape. Microglial proliferation was also facilitated by LPS but not by poly(I:C). In addition, LPS and poly(I:C) modulated microglial chemotaxis and phagocytosis differently. Furthermore, genome-wide analysis provided gene-level support to these functional differences, which may be associated with NF-κb and type I interferon pathways. Last, LPS- and poly(I:C)-activated microglia mediated neurotoxicity in a co-culture system. This study extends our understanding of TLR roles in microglia and provides insights into selecting proper inflammatory microglial models, which may facilitate identification of new targets for therapeutic application.


Assuntos
Lipopolissacarídeos/farmacologia , Microglia/efeitos dos fármacos , Poli I-C/farmacologia , Animais , Células Cultivadas , Quimiotaxia/efeitos dos fármacos , Quimiotaxia/imunologia , Técnicas de Cocultura , Feminino , Interferon Tipo I/metabolismo , Camundongos , Microglia/imunologia , NF-kappa B/metabolismo , Neurônios , Fagocitose/efeitos dos fármacos , Fagocitose/imunologia , Cultura Primária de Células , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Receptor 3 Toll-Like/agonistas , Receptor 3 Toll-Like/metabolismo , Receptor 4 Toll-Like/agonistas , Receptor 4 Toll-Like/metabolismo
20.
Sci Rep ; 11(1): 11298, 2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-34050236

RESUMO

Berberine is a well-known quaternary ammonium salt that is usually found in the roots of such plants as Phellodendron amurense and Coptis chinensis. However, the effects of berberine on double-stranded RNA (dsRNA)-induced macrophages have not been fully reported. In this study, we examined the anti-inflammatory effects of berberine on dsRNA [polyinosinic-polycytidylic acid; poly I:C]-induced macrophages. Levels of nitric oxide (NO), Prostaglandin E2 (PGE2), first apoptosis signal receptor (Fas; CD95), cytokines, intracellular calcium, phosphorylated I-kappa-B-alpha (IkB-α), phosphorylated p38 mitogen-activated protein kinase (MAPK), phosphorylated ERK1/2, phosphorylated signal transducer and activated transcription 3 (STAT3), and mRNA expression of inflammatory genes in poly I:C-induced RAW 264.7 mouse macrophages were evaluated. Berberine significantly inhibited the production of NO, PGE2, Fas, GM-CSF, LIF, LIX, RANTES, and MIP-2 as well as calcium release in poly I:C-induced RAW 264.7 cells at concentrations of up to 50 µM. Berberine also significantly inhibited the phosphorylation of p38 MAPK, ERK1/2, IkB-α, and STAT3 in poly I:C-induced RAW 264.7 cells. Additionally, berberine significantly decreased the mRNA expressions of Chop (GADD153), Stat1, Stat3, and Fas in poly I:C-induced RAW 264.7 cells. Taken together, berberine has anti-inflammatory properties related to its inhibition of NO, PGE2, Fas, GM-CSF, LIF, LIX, RANTES, and MIP-2 in dsRNA-induced macrophages via the endoplasmic reticulum stress-related calcium-CHOP/STAT pathway.


Assuntos
Berberina/química , Berberina/farmacologia , Macrófagos/metabolismo , Animais , Anti-Inflamatórios/imunologia , Anti-Inflamatórios/metabolismo , Anti-Inflamatórios/farmacologia , Berberina/metabolismo , Cálcio/metabolismo , Citocinas/metabolismo , Inflamação/metabolismo , Macrófagos/efeitos dos fármacos , Camundongos , Óxido Nítrico/metabolismo , Poli I-C/farmacologia , Células RAW 264.7 , Fatores de Transcrição STAT/metabolismo , Fator de Transcrição STAT1 , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição CHOP/metabolismo
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