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Frontiers in Immunology 2023Interferon (IFN) responses are critical in the resolution of viral infections and are actively targeted by many viruses. They also play a role in inducing protective...
BACKGROUND
Interferon (IFN) responses are critical in the resolution of viral infections and are actively targeted by many viruses. They also play a role in inducing protective responses after vaccination and have been successfully tested as vaccine adjuvants. IFN responses are well conserved and function very similar in teleosts and mammals. Like in mammals, IFN responses in piscine cells are initiated by intracellular detection of the viral infection by different pattern recognition receptors. Upon the recognition of viral components, IFN responses are rapidly induced to combat the infection. However, many viruses may still replicate and be able to inhibit or circumvent the IFN response by different means.
METHODS
By employing CRISPR Cas9 technology, we have disrupted proteins that are central for IFN signaling in the salmonid cell line CHSE-214. We successfully generated KO clones for the mitochondrial antiviral signaling protein MAVS, the transcription factors IRF3 and IRF7-1, as well as a double KO for IRF7-1/3 using an optimized protocol for delivery of CRISPR-Cas ribonucleoproteins through nucleofection.
RESULTS
We found that MAVS and IRF3 KOs inhibited IFN and IFN-stimulated gene induction after intracellular poly I:C stimulation as determined through gene expression and promoter activation assays. In contrast, the IRF7-1 KO had no clear effect. This shows that MAVS and IRF3 are essential for initiation of intracellular RNA-induced IFN responses in CHSE-214 cells. To elucidate viral interference with IFN induction pathways, the KOs were infected with Salmon alphavirus 3 (SAV3) and infectious pancreatic necrosis virus (IPNV). SAV3 infection in control and IRF7-1 KO cells yielded similar titers and no cytopathic effect, while IRF3 and MAVS KOs presented with severe cytopathic effect and increased titers 6 days after SAV 3 infection. In contrast, IPNV yields were reduced in IRF3 and MAVS KOs, suggesting a dependency on interactions between viral proteins and pattern recognition receptor signaling components during viral replication.
CONCLUSION
Aside from more insight in this signaling in salmonids, our results indicate a possible method to increase viral titers in salmonid cells.
Topics: Animals; Salmonidae; CRISPR-Cas Systems; Signal Transduction; Cell Line; Salmon; Infectious pancreatic necrosis virus; Mammals
PubMed: 37588594
DOI: 10.3389/fimmu.2023.1214912 -
Frontiers in Immunology 2024NOD1 and NOD2 as two representative members of nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family play important roles in antimicrobial immunity....
NOD1 and NOD2 as two representative members of nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family play important roles in antimicrobial immunity. However, transcription mechanism of and and their signal circle are less understood in teleost fish. In this study, with the cloning of and in Chinese perch, the interaction between NOD1, NOD2, and CARD9 and RIPK2 were revealed through coimmunoprecipitation and immunofluorescence assays. The overexpression of NOD1, NOD2, RIPK2 and CARD9 induced significantly the promoter activity of NF-κB, IFNh and IFNc. Furthermore, it was found that and were induced by poly(I:C), type I IFNs, RLR and even NOD1/NOD2 themselves through the ISRE site of their proximal promoters. It is thus indicated that and can be classified also as ISGs due to the presence of ISRE in their proximal promoter, and their expression can be mechanistically controlled through PRR pathway as well as through IFN signaling in antiviral immune response.
Topics: Animals; Nod1 Signaling Adaptor Protein; Receptor-Interacting Protein Serine-Threonine Kinase 2; Nod2 Signaling Adaptor Protein; Signal Transduction; Fish Proteins; Perches; Interferons; Promoter Regions, Genetic; Transcription, Genetic; Immunity, Innate; Protein Binding
PubMed: 38715616
DOI: 10.3389/fimmu.2024.1374368 -
Journal of Neuroinflammation May 2024Maternal inflammation during gestation is associated with a later diagnosis of neurodevelopmental disorders including autism spectrum disorder (ASD). However, the...
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.
Topics: Female; Animals; Pregnancy; Male; Cytokines; Mice; Brain; Placenta; Mice, Inbred C57BL; Prenatal Exposure Delayed Effects; Neurodevelopmental Disorders; Sex Characteristics; Poly I-C; Transcriptome; Disease Models, Animal; Fetus
PubMed: 38715090
DOI: 10.1186/s12974-024-03106-7 -
Frontiers in Cell and Developmental... 2023Mesenchymal stromal cells (MSC) are envisioned as a potential cellular vehicle for targeted cancer therapies due to their tumor tropism and immune permissiveness. An...
Mesenchymal stromal cells (MSC) are envisioned as a potential cellular vehicle for targeted cancer therapies due to their tumor tropism and immune permissiveness. An obstacle in their use is the duality in their interactions within tumors, rendering them pro-tumorigenic or anti-tumorigenic, in a context dependent manner. MSC preconditioning, or priming, has been proposed as a strategy for directing the effector properties of MSC at tumor sites. We primed human MSC derived from adipose tissues (ASC), a clinically advantageous MSC source, utilizing toll-like receptor agonists. Subsequently, we explored the consequences in tumor progression and transcriptome upon the interaction of tumor cells with primed or unprimed ASC in an model of prostate cancer, the second most common cancer and second leading cause of cancer related death in men in the USA. In the studied model, poly I:C-primed ASC were found to significantly accelerate tumor growth progression. And while unprimed and LPS-primed ASC did not exert a significant effect on tumor growth at the macroscopic level, gene expression analyses suggested that all treatments promoted distinct modulatory effects in the tumor microenvironment, including altered modulation of angiogenesis, and immune response processes. However, the effects resulting from the collective interaction across these processes must be sufficiently skewed in a pro-tumorigenic or anti-tumorigenic direction for evidence of tumor progression modulation to be detectable at the macroscopic level. Our study highlights potential MSC-tumor microenvironment interactions that may be leveraged and should be considered in the development of cancer therapeutics utilizing MSC.
PubMed: 38078010
DOI: 10.3389/fcell.2023.1145421 -
Poultry Science Oct 2023Innate immunity, as an organism's first line of defense, plays a crucial role in rapidly responding to and protecting the body against invading pathogens. As a cytosolic...
Innate immunity, as an organism's first line of defense, plays a crucial role in rapidly responding to and protecting the body against invading pathogens. As a cytosolic RNA sensor for viral infections, including infections caused by influenza virus, the innate immune system in chickens has 2 major pathogen-recognition receptors (PRRs): Toll-like receptor 3 (TLR3) and melanoma differentiation-associated protein 5 (MDA5). The signaling pathways activated by PRRs are complex, systemic processes that underlie the response to foreign molecules. In this study, we investigated the interactions among MDA5, mitochondrial antiviral signaling protein (MAVS), and stimulator of interferon genes (STING) signaling in chicken cells. To exclude the effects of TLR3, we transfected the clustered regularly interspaced palindromic repeats/CRISPR-associated protein 9 (CRISPR-Cas9) expression vector and TLR3-targeted gRNA plasmid into chicken DF-1 cells. We selected TLR3-knockout (KO) cell line and sequentially, we established 2 double-KO cell lines: TLR3-MAVS KO and TLR3-STING KO. After treatment with polyinosinic:polycytidylic acid (poly(I:C)), type I interferon (IFN), IFN-stimulated gene, and antiviral gene (IFN regulatory factor 7, IFNβ, Mx1, and protein kinase R1) expression was not completely activated in TLR3-MAVS KO cells, whereas it was consistently upregulated in wild-type and TLR3-STING KO DF-1 cells. These results suggest that STING is not an intermediator between MDA5 and MAVS; moreover, it does not directly interact with MDA5 during innate immune activation in chicken DF-1 cells.
Topics: Animals; Interferon-Induced Helicase, IFIH1; Chickens; Toll-Like Receptor 3; Signal Transduction; Immunity, Innate; Antiviral Agents
PubMed: 37473519
DOI: 10.1016/j.psj.2023.102913 -
Pharmaceutics Jul 2023Macrophages (MΦs) in their pro-inflammatory state (M1) suppress tumour growth, while tumour-associated MΦs (TAMs) can promote tumour progression. The aim of this study...
Macrophages (MΦs) in their pro-inflammatory state (M1) suppress tumour growth, while tumour-associated MΦs (TAMs) can promote tumour progression. The aim of this study was to test the hypothesis that targeted delivery of the immune activator poly(I:C) in aspherical silica microrods (µRs) can repolarize TAMs into M1-like cells. µRs (10 µm × 3 µm) were manufactured from silica nanoparticles and stabilized with dextran sulphate and polyethyleneimine. The THP-1 cell line, differentiated into MΦs, and primary human monocyte-derived MΦs (HMDMs) were treated with tumour-cell-conditioned medium (A549), but only HMDMs could be polarized towards TAMs. Flow cytometry and microscopy revealed elevated uptake of µRs by TAMs compared to non-polarized HMDMs. Flow cytometry and qPCR studies on polarization markers showed desirable effects of poly(I:C)-loaded MPs towards an M1 polarization. However, unloaded µRs also showed distinct actions, which were not induced by bacterial contaminations. Reporter cell assays showed that µRs induce the secretion of the inflammatory cytokine IL-1β. Macrophages from knockout mice showed that µRs in concentrations as low as 0.5 µR per cell can activate the inflammasome and induce cell death. In conclusion, our data show that µRs, even if unloaded, can induce inflammasome activation and cell death in low concentrations.
PubMed: 37514081
DOI: 10.3390/pharmaceutics15071895 -
International Journal of Molecular... Nov 2023Acute respiratory distress syndrome (ARDS) has no specific and effective treatment, and there is an urgent need to understand its pathogenesis. Therefore, based on the...
A Disintegrin and Metalloproteinase with Thrombospondin Motifs 4 Regulates Pulmonary Vascular Hyperpermeability through Destruction of Glycocalyx in Acute Respiratory Distress Syndrome.
Acute respiratory distress syndrome (ARDS) has no specific and effective treatment, and there is an urgent need to understand its pathogenesis. Therefore, based on the hypothesis that molecules whose expression is upregulated in injured pulmonary vascular endothelial cells (VECs) are involved in the pathogenesis of ARDS, we conducted a study to elucidate the molecular mechanisms and identify target factors for treatment. Primary human lung microvascular endothelial cells (HMVEC-Ls) were stimulated with lipopolysaccharide (LPS) or poly (I:C) and analyzed via a microarray to identify target genes for ARDS. We found that a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4) was induced in murine lung VECs in an LPS-mediated ARDS model. Elevated ADAMTS4 was also observed by the immunostaining of lung samples from ARDS patients. The suppression of ADAMTS4 by siRNA in VECs ameliorated LPS-stimulated vascular permeability. The impairment of the cell surface expression of syndecan-1, a marker of the glycocalyx that is an extracellular matrix involved in vascular permeability, was dramatically inhibited by ADAMTS4 suppression. In addition, the suppression of ADAMTS4 protected against LPS-induced reductions in syndecan-1 and the adherens junction protein vascular endothelial cadherin. These results suggest that ADAMTS4 regulates VEC permeability in ARDS and may be a predictive marker and therapeutic target for ARDS.
Topics: Humans; Animals; Mice; Endothelial Cells; Glycocalyx; Disintegrins; Syndecan-1; Lipopolysaccharides; Respiratory Distress Syndrome; Lung; Thrombospondins; Metalloproteases
PubMed: 38003418
DOI: 10.3390/ijms242216230 -
The Journal of Biological Chemistry Sep 2023Exosomes, extracellular vesicles (EVs) produced within cells, mediate both the disposal of intracellular waste and communication with distant cells, and they are...
Exosomes, extracellular vesicles (EVs) produced within cells, mediate both the disposal of intracellular waste and communication with distant cells, and they are involved in a variety of disease processes. Although disease modifications of exosome cargos have been well studied, it has been poorly investigated how disease processes, such as endoplasmic reticulum (ER) stress, affect EV production. We previously reported that adiponectin, an adipocyte-secreted salutary factor, increases systemic exosome levels through T-cadherin-mediated enhancement of exosome biogenesis. In the present study, we demonstrated that adiponectin/T-cadherin-dependent EV production was susceptible to ER stress and that low-dose tunicamycin significantly reduced EV production in the presence, but not in the absence, of adiponectin. Moreover, pharmacological or genetic activation of inositol-requiring enzyme 1α, a central regulator of ER stress, downregulated T-cadherin at the mRNA and protein levels as well as attenuated EV production. In addition, adiponectin/T-cadherin-independent EV production was attenuated under ER stress conditions. Repeated administration of tunicamycin to mice decreased circulating small EVs without decreasing tissue T-cadherin expression. Mechanistically, inositol-requiring enzyme 1α activation by silencing of the X-box binding protein 1 transcription factor upregulated the canonical interferon pathway and decreased EV production. The interferon pathway, when it was activated by polyinosinic-polycytidylic acid, also significantly attenuated EV production. Thus, we concluded that ER stress decreases exosome production through adiponectin/T-cadherin-dependent and -independent pathways.
Topics: Animals; Mice; Adiponectin; Cadherins; Endoplasmic Reticulum Stress; Exosomes; Inositol; Interferons; Poly I-C; Tunicamycin
PubMed: 37524131
DOI: 10.1016/j.jbc.2023.105114 -
IScience Aug 2023Oxylipins are major immunomodulating mediators, yet studies of inflammation focus mainly on cytokines. Here, using a standardized whole-blood stimulation system, we...
Oxylipins are major immunomodulating mediators, yet studies of inflammation focus mainly on cytokines. Here, using a standardized whole-blood stimulation system, we characterized the oxylipin-driven inflammatory responses to various stimuli and their relationships with cytokine responses. We performed a pilot study in 25 healthy individuals using 6 different stimuli: 2 bacterial stimuli (LPS and live BCG), 2 viral stimuli (vaccine-grade poly I:C and live H1N1 attenuated influenza), an enterotoxin superantigen and a Null control. All stimuli induced a strong production of oxylipins but most importantly, bacterial, viral, and T cell immune responses show distinct oxylipin signatures. Integration of the oxylipin and cytokine responses for each condition revealed new immune networks improving our understanding of inflammation regulation. Finally, the oxylipin responses and oxylipin-cytokine networks were compared in patients with active tuberculosis or with latent infection. This revealed different responses to BCG but not LPS stimulation highlighting new regulatory pathways for further investigations.
PubMed: 37575177
DOI: 10.1016/j.isci.2023.107422 -
International Journal of Molecular... Aug 2023Long-term treatments for inflammatory skin diseases like atopic dermatitis or eczema can cause adverse effects. Super Protein Multifunction (SPM) was investigated as a... (Randomized Controlled Trial)
Randomized Controlled Trial
Long-term treatments for inflammatory skin diseases like atopic dermatitis or eczema can cause adverse effects. Super Protein Multifunction (SPM) was investigated as a potential treatment for managing skin inflammation by monitoring the expression of pro-inflammatory cytokines induced using LPS and poly(I:C)/TNFα in HaCaT keratinocytes and Hs27 fibroblasts as measured via RT-PCR. SPM solution was also assessed for its effect on cytokine release, measured using ELISA, in a UVB-irradiated 3D human skin model. To evaluate the efficiency of SPM, 20 patients with mild eczematous skin were randomized to receive SPM or vehicle twice a day for three weeks in a double-blind controlled trial. In vitro studies showed SPM inhibited inflammation-induced IL-1β, IL-6, IL-33, IL-1α, TSLP, and TNFα expression or release. In the clinical study, the SPM group showed significant improvements in the IGA, PA, and DLQI scores compared to the vehicle group. Neither group showed significant differences in VAS (pruritus). Histological analysis showed reduced stratum corneum thickness and inflammatory cell infiltration. The results suggest that SPM may reduce inflammation in individuals with chronic eczematous skin.
Topics: Humans; Tumor Necrosis Factor-alpha; Eczema; Skin; Inflammation; Pruritus; Cytokines; Excipients
PubMed: 37629159
DOI: 10.3390/ijms241612979