ABSTRACT
Aicardi-Goutières syndrome (AGS) is an autoinflammatory disease characterized by aberrant interferon (IFN)-α production. The major cause of morbidity in AGS is brain disease, yet the primary source and target of neurotoxic IFN-α remain unclear. Here, we demonstrated that the brain was the primary source of neurotoxic IFN-α in AGS and confirmed the neurotoxicity of intracerebral IFN-α using astrocyte-driven Ifna1 misexpression in mice. Using single-cell RNA sequencing, we demonstrated that intracerebral IFN-α-activated receptor (IFNAR) signaling within cerebral endothelial cells caused a distinctive cerebral small vessel disease similar to that observed in individuals with AGS. Magnetic resonance imaging (MRI) and single-molecule ELISA revealed that central and not peripheral IFN-α was the primary determinant of microvascular disease in humans. Ablation of endothelial Ifnar1 in mice rescued microvascular disease, stopped the development of diffuse brain disease, and prolonged lifespan. These results identify the cerebral microvasculature as a primary mediator of IFN-α neurotoxicity in AGS, representing an accessible target for therapeutic intervention.
Subject(s)
Brain , Interferon-alpha , Microvessels , Nervous System Malformations , Receptor, Interferon alpha-beta , Animals , Humans , Mice , Interferon-alpha/metabolism , Brain/metabolism , Brain/pathology , Receptor, Interferon alpha-beta/metabolism , Receptor, Interferon alpha-beta/genetics , Microvessels/pathology , Nervous System Malformations/genetics , Autoimmune Diseases of the Nervous System/immunology , Endothelial Cells/metabolism , Mice, Knockout , Male , Female , Signal Transduction , Mice, Inbred C57BL , Astrocytes/metabolism , Disease Models, AnimalABSTRACT
NKp44 is a human receptor originally found on activated NK cells, group 1 and group 3 innate lymphoid cells that binds dimers of platelet-derived growth factor D (PDGF-DD). NKp44 is also expressed on tissue plasmacytoid dendritic cells (PDCs), but NKp44-PDGF-DD interaction on PDCs remains unstudied. Engagement of NKp44 with PDGF-DD in vitro enhanced PDC secretion of IFN-α, TNF, and IL-6 in response to the TLR9 ligand CpG-ODN, but not TLR7/8 ligands. In tissues, PDCs were found in close contact with PDGF-DD-expressing cells in the high endothelial venules and epithelium of tonsils, melanomas, and skin lesions infected with Molluscum contagiosum. Recombinant PDGF-DD enhanced the serum IFN-α response to systemic HSV-1 infection in a humanized mouse model. We conclude that NKp44 integrates with TLR9 signaling to enhance PDC cytokine production. These findings may have bearings for immune responses to TLR9-based adjuvants, therapy for tumors expressing PDGF-DD, and infections with DNA viruses that induce PDGF-DD expression to enhance viral spread.
Subject(s)
Immunity, Innate , Toll-Like Receptor 9 , Animals , Mice , Humans , Toll-Like Receptor 9/metabolism , Interferon-alpha/metabolism , Dendritic Cells , Killer Cells, NaturalABSTRACT
Enterovirus 71 (EV71) is a significant causative agent of hand, foot, and mouth disease, with potential serious neurologic complications or fatal outcomes. The lack of effective treatments for EV71 infection is attributed to its elusive pathogenicity. Our study reveals that human plasmacytoid dendritic cells (pDCs), the main type I IFN-producing cells, selectively express scavenger receptor class B, member 2 (SCARB2) and P-selectin glycoprotein ligand 1 (PSGL-1), crucial cellular receptors for EV71. Some strains of EV71 can replicate within pDCs and stimulate IFN-α production. The activation of pDCs by EV71 is hindered by Abs to PSGL-1 and soluble PSGL-1, whereas Abs to SCARB2 and soluble SCARB2 have a less pronounced effect. Our data suggest that only strains binding to PSGL-1, more commonly found in severe cases, can replicate in pDCs and induce IFN-α secretion, highlighting the importance of PSGL-1 in these processes. Furthermore, IFN-α secretion by pDCs can be triggered by EV71 or UV-inactivated EV71 virions, indicating that productive infection is not necessary for pDC activation. These findings provide new insights into the interaction between EV71 and pDCs, suggesting that pDC activation could potentially mitigate the severity of EV71-related diseases.
Subject(s)
Dendritic Cells , Enterovirus A, Human , Interferon-alpha , Lysosomal Membrane Proteins , Membrane Glycoproteins , Dendritic Cells/immunology , Dendritic Cells/virology , Humans , Enterovirus A, Human/immunology , Enterovirus A, Human/physiology , Membrane Glycoproteins/metabolism , Lysosomal Membrane Proteins/metabolism , Lysosomal Membrane Proteins/immunology , Interferon-alpha/metabolism , Interferon-alpha/immunology , Receptors, Scavenger/metabolism , Enterovirus Infections/immunology , Enterovirus Infections/virology , Virus ReplicationABSTRACT
Previous reports have shown that IL-6 and IFN-⺠induce distinct transcriptomic and morphological changes in microglia. Here, we demonstrate that IL-6 increases tissue surveillance, migration and phagocytosis in primary murine microglia, whereas IFN-⺠inhibits these functions. Our results provide a crucial link between transcriptome and function. It holds the potential to serve as the foundation for future studies aimed at identifying therapeutic targets for cytokine-mediated neuroinflammatory diseases.
Subject(s)
Interferon-alpha , Interleukin-6 , Microglia , Animals , Mice , Cell Movement/drug effects , Interferon-alpha/metabolism , Interleukin-6/metabolism , Mice, Inbred C57BL , Microglia/metabolism , Microglia/drug effects , Phagocytosis/physiology , Phagocytosis/drug effectsABSTRACT
BACKGROUND: Lytic Epstein-Barr virus (EBV) infection plays a major role in the pathogenesis of nasopharyngeal carcinoma (NPC). For patients with recurrent or metastatic NPC and resistant to conventional therapies, adoptive cell therapy using EBV-specific cytotoxic T cells (EBV-CTLs) is a promising option. However, the long production period (around 3 to 4 weeks) and low EBV-CTL purity (approximately 40% of total CD8 T cells) in the cell product limits the application of EBV-CTLs in clinics. Thus, this study aimed to establish a protocol for the rapid production of EBV-CTLs. METHODS: By culturing peripheral blood mononuclear cells (PBMCs) from EBV-seropositive donors with EBV-specific peptides and interleukin (IL)-2, IL-15, and interferon α (IFN-α) for 9 days, we identified that IL-15 can enhance IL-2-mediated CTL activation and significantly increase the yield of CTLs. RESULTS: When IFN-α was used in IL-2/IL-15-mediated CTL production from days 0 to 6, the productivity of EBV-CTLs and EBV-specific cytotoxicity significantly were reinforced relative to EBV-CTLs from IL-2/IL-15 treatment. Additionally, IFN-α-induced production improvement of virus-specific CTLs was not only the case for EBV-CTLs but also for cytomegalovirus-specific CTLs. CONCLUSION: We established a novel protocol to rapidly expand highly pure EBV-CTLs from PBMCs, which can produce EBV-CTLs in 9 days and does not require feeder cells during cultivation.
Subject(s)
Herpesvirus 4, Human , T-Lymphocytes, Cytotoxic , Humans , T-Lymphocytes, Cytotoxic/immunology , Herpesvirus 4, Human/immunology , Epstein-Barr Virus Infections/immunology , Epstein-Barr Virus Infections/virology , Interleukin-2/metabolism , Interleukin-2/pharmacology , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/virology , Interleukin-15/metabolism , Interferon-alpha/metabolism , Cytotoxicity, Immunologic , Nasopharyngeal Carcinoma/virology , Nasopharyngeal Carcinoma/immunology , Nasopharyngeal Carcinoma/pathology , Nasopharyngeal Neoplasms/immunology , Nasopharyngeal Neoplasms/virology , Nasopharyngeal Neoplasms/pathology , Lymphocyte Activation/immunology , Immunotherapy, Adoptive/methodsABSTRACT
BACKGROUND: Hyperactive neutrophil extracellular traps (NETs) formation plays a crucial role in active severe systemic lupus erythematosus (SLE). However, what triggers the imbalance in dysregulated NETs formation in SLE is elusive. Transfer RNA-derived small RNAs (tsRNAs) are novel non-coding RNAs, which participate in various cellular processes. We explore the role of tsRNAs on NETs formation in SLE. METHODS: We analyzed the levels of NETs DNA and platelet-derived extracellular vesicles (pEVs) from 50 SLE patients and 20 healthy control subjects. The effects of pEVs on NETs formation were evaluated by using immunofluorescence assay and myeloperoxidase-DNA PicoGreen assay. The regulatory mechanism of pEVs on NETs formation and inflammatory cytokines production were investigated using an in vitro cell-based assay. RESULTS: Increased circulating NETs DNA and pEVs were shown in SLE patients and were associated with disease activity (P < 0.005). We demonstrated that SLE patient-derived immune complexes (ICs) induced platelet activation, followed by pEVs release. ICs-triggered NETs formation was significantly enhanced in the presence of pEVs through Toll-like receptor (TLR) 8 activation. Increased levels of tRF-His-GTG-1 in pEVs and neutrophils of SLE patients were associated with disease activity. tRF-His-GTG-1 interacted with TLR8 to prime p47phox phosphorylation in neutrophils, resulting in reactive oxygen species production and NETs formation. Additionally, tRF-His-GTG-1 modulated NF-κB and IRF7 activation in neutrophils upon TLR8 engagement, resulting IL-1ß, IL-8, and interferon-α upregulation, respectively. CONCLUSIONS: The level of tRF-His-GTG-1 was positively correlated with NETs formation in SLE patients; tRF-His-GTG-1 inhibitor could efficiently suppress ICs-triggered NETs formation/hyperactivation, which may become a potential therapeutic target.
Neutrophils and platelets are key members in the immunopathogenesis of SLE. EVs play a key role in intercellular communication. Abnormal NETs formation promotes vascular complications and organ damage in SLE patients. tsRNA is a novel regulatory small non-coding RNA and participates in diverse pathological processes. Herein, we showed that SLE patient-derived ICs activates platelets directly, followed by intracellular tRF-His-GTG-1 upregulation, which is loaded into pEVs. The pEV-carried tRF-His-GTG-1 could interact with TLR8 in neutrophils, followed by activation of the downstream signaling pathway, including p47phox-NOX2-ROS, which causes NETs enhancement, while IRF7 promotes the expression of IFN-α. The tRF-His-GTG-1 inhibitor could suppress efficiently SLE ICs-induced NETs formation and pEVs primed NETs enhancement. This study offers new molecular machinery to explain the association between the platelets-derived tsRNAs, pEVs, and hyperactive NETs formation in lupus. tRF-His-GTG-1 may serve as a potential therapeutic target and help to advance our understanding of tsRNAs in SLE pathogenesis.
Subject(s)
Extracellular Traps , Extracellular Vesicles , Interferon-alpha , Lupus Erythematosus, Systemic , Adult , Female , Humans , Male , Middle Aged , Blood Platelets/metabolism , Extracellular Traps/metabolism , Extracellular Vesicles/metabolism , Interferon-alpha/metabolism , Lupus Erythematosus, Systemic/metabolism , Lupus Erythematosus, Systemic/genetics , Neutrophils/metabolism , Toll-Like Receptor 8/metabolism , Toll-Like Receptor 8/genetics , RNA, Transfer/chemistry , RNA, Transfer/metabolismABSTRACT
OBJECTIVE: This study aimed to investigate the possible role of granulin (GRN) in activating the TLR9-IFN-α pathway in renal tubular epithelial cells (RTECs) and explore clues that RTECs regulate the micro-environment of inflammatory response in lupus nephritis (LN). METHODS: Renal sections from 57 LN patients and 30 non-LN patients were sampled for histological study, and GRN overexpression RTECs were applied for cytological study. RESULTS: In the histological study, GRN is highly expressed in LN RTECs with tubulointerstitial inflammation (TII) and well co-localized with TLR9. ROC analysis suggested a potential relationship between GRN expression in RTECs and therapeutic response. Moreover, IFN-α also highly expressed in LN RTECs with TII, and the intensity of IFN-α is positively correlated with the co-localization intensity of GRN and TLR9. In the cytological study, LN serum, especially serum from LN with TII, activates the expression of TLR9 in RTECs, and GRN engages the interaction of TLR9 to activate the expression of IFN-α in RTECs. While TLR9 inhibitors can suppress the expression of IFN-α in RTECs, the degree of inhibition is dose-dependent. CONCLUSION: The expression of GRN in RTECs is associated with interstitial inflammation and therapeutic response. GRN may mediate the activation of the TLR9-IFN-α pathway in RTECs and involve in the micro-environment of inflammatory response in LN.
Subject(s)
Granulins , Lupus Erythematosus, Systemic , Lupus Nephritis , Humans , Granulins/metabolism , Inflammation/metabolism , Interferon-alpha/metabolism , Kidney/pathology , Lupus Erythematosus, Systemic/pathology , Lupus Nephritis/pathology , Toll-Like Receptor 9/metabolismABSTRACT
BACKGROUND: Type I interferons (IFN-I)-a group of cytokines with immunomodulatory, antiproliferative, and antiviral properties-are widely used as therapeutics for various cancers and viral diseases. Since IFNs are proteins, they are highly susceptible to degradation by proteases and by hydrolysis in the strong acid environment of the stomach, and they are therefore administered parenterally. In this study, we examined whether the intestinal bacterium, enteropathogenic Escherichia coli (EPEC), can be exploited for oral delivery of IFN-Is. EPEC survives the harsh conditions of the stomach and, upon reaching the small intestine, expresses a type III secretion system (T3SS) that is used to translocate effector proteins across the bacterial envelope into the eukaryotic host cells. RESULTS: In this study, we developed an attenuated EPEC strain that cannot colonize the host but can secrete functional human IFNα2 variant through the T3SS. We found that this bacteria-secreted IFN exhibited antiproliferative and antiviral activities similar to commercially available IFN. CONCLUSION: These findings present a potential novel approach for the oral delivery of IFN via secreting bacteria.
Subject(s)
Enteropathogenic Escherichia coli , Type III Secretion Systems , Enteropathogenic Escherichia coli/metabolism , Humans , Type III Secretion Systems/metabolism , Interferon-alpha/metabolism , Antiviral Agents/pharmacology , Antiviral Agents/metabolism , Interferon alpha-2/metabolism , Cell Proliferation/drug effectsABSTRACT
Hypertrophic scars (HTS) develop from an excessive synthesis of structural proteins like collagen and a decreased expression of proteoglycans such as decorin. Previous research has demonstrated that decorin expression is significantly down-regulated in HTS, deep dermal tissue, and thermally injured tissue, reducing its ability to regulate pro-fibrotic transforming growth factor-beta 1 (TGF-ß1) and normal fibrillogenesis. However, treatment of HTS fibroblasts with interferon-alpha 2b (IFN-α2b) has been shown to reduce excessive collagen synthesis and improve HTS by reducing serum TGF-ß1 levels. The expression of decorin isoforms in HTS is currently unknown and the effects of TGF-ß1 and IFN-α2b on decorin, decorin isoform expression and type 1 collagen are of great interest to our group. Dermal fibroblasts were treated with TGF-ß1 and/or IFN-α2b, for 48 h. The expression and secretion of decorin, decorin isoforms and type 1 collagen were quantified with reverse transcription-quantitative polymerase chain reaction, immunofluorescence staining and enzyme-linked immunosorbent assays. The mRNA expression of decorin and each isoform was significantly reduced in HTS fibroblasts relative to normal skin. TGF-ß1 decreased the mRNA expression of decorin and decorin isoforms, whereas IFN-α2b showed the opposite effect. IFN-α2b significantly inhibited TGF-ß1's effect on the mRNA expression of type I collagen alpha 1 in papillary dermal fibroblasts and overall showed relative effects of inhibiting TGF-ß1. These data support that a further investigation into the structural and functional roles of decorin isoforms in HTS pathogenesis is warranted and that IFN-α2b is an important agent in reducing fibrotic outcomes.
Subject(s)
Cicatrix, Hypertrophic , Collagen Type I , Interferon alpha-2 , Humans , Cells, Cultured , Cicatrix, Hypertrophic/pathology , Collagen/metabolism , Collagen Type I/metabolism , Decorin/metabolism , Fibroblasts/metabolism , Interferon-alpha/pharmacology , Interferon-alpha/metabolism , Protein Isoforms/metabolism , Protein Isoforms/pharmacology , RNA, Messenger/metabolism , Transforming Growth Factor beta1/metabolism , Wound Healing/physiologyABSTRACT
Enterovirus 71 (EV71) is a neurotropic enterovirus associated with hand, foot, and mouth disease (HFMD) fatalities. In this study, we investigated the impact of EV71 on plasmacytoid dendritic cells (pDCs) and CD4+ T cells. The results showed that pDCs were promptly activated, secreting interferon (IFN)-α and inducing CD4+ T cell proliferation and differentiation during early EV71 infection. This initiated adaptive immune responses and promoted proinflammatory cytokine production by CD4+ T cells. Over time, viral nucleic acids and proteins were synthesized in pDCs and CD4+ T cells. Concurrently, the cholinergic anti-inflammatory pathway (CAP) was activated, exhibiting an anti-inflammatory role. With constant viral stimulation, pDCs and CD4+ T cells showed reduced differentiation and cytokine secretion. Defects in pDCs were identified as a key factor in CD4+ T cell tolerance. CAP had a more significant regulatory effect on CD4+ T cells than on pDCs and was capable of inhibiting inflammation in these cells.
Subject(s)
Enterovirus A, Human , Enterovirus Infections , Humans , Neuroimmunomodulation , Up-Regulation , Interferon-alpha/metabolism , Cell Differentiation , Enterovirus Infections/metabolism , CD4-Positive T-Lymphocytes , Dendritic CellsABSTRACT
Triple motif protein 21 (TRIM21) has an antiviral function that inhibits various viral infections. However, its role in the progress of influenza A virus (IAV) infection is unclear. In this study, we investigated the role and molecular mechanism of TRIM21 in IAV infection. RT-qPCR was used to determine the level of TRIM21 mRNA, and ELISA was used to measure the levels of IFN-α, IFN-ß, IL-6, and TNF-α. The levels of the TRIM21, NP, TBK1, IRF3, p-TBK1, and p-IRF3 proteins were estimated by Western blot. The results showed that, after IAV infection, TRIM21 was upregulated in clinical patient serum and A549 cells, and this was correlated with the IFN response. Overexpression of TRIM21 reduced IAV replication and transcription in in vitro cell experiments. TRIM21 also increased IFN-α and IFN-ß levels and decreased IL-6 and TNF-α levels in A549 cells. In addition, overexpression of TRIM21 inhibited IAV-induced apoptosis. Further experiments demonstrated that TBK1-IRF3 signaling was activated by TRIM21 and was involved in the inhibitory effect of TRIM21 on virus replication. In summary, our study suggests that TRIM21 inhibits viral replication by activating the TBK1-IRF3 signaling pathway, further inhibiting the infection process of IAV.
Subject(s)
Influenza A Virus, H1N1 Subtype , Influenza A virus , Influenza, Human , Humans , A549 Cells , Inflammation , Influenza A virus/metabolism , Influenza A Virus, H1N1 Subtype/metabolism , Influenza, Human/genetics , Interferon Regulatory Factor-3/genetics , Interferon Regulatory Factor-3/metabolism , Interferon-alpha/metabolism , Interleukin-6/genetics , Interleukin-6/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Signal Transduction/physiology , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Human interferon (hINF) alpha 2b is clinically important pharmaceutical product included in combinatory therapy against chronic hepatitis C and B and complex therapy against several cancer diseases. Here, we created the genetic constructions, based on genome elements of potato virus X (PVX), carrying the infα2b gene for transient expression in plant cells. The created plasmid vector constructions were tested through Agrobacterium-mediated transient gene expression method in two plant species-Nicotiana benthamiana and Ocimum basilicum (sweet basil). Production of recombinant hINF alpha 2b was more efficient in N. benthamiana than that in O. basilicum plants. The average yield of hINF alpha 2b produced in N. benthamiana plants was 0.56 mg/g of fresh leaf weight (FW) or 6% of the total soluble cell proteins (TSP). The maximal level reached up to 1.2 mg/g FW or 9% TSP. We estimated that about 0.67 mg of hINF can be obtained from one N. benthamiana plant. The yield of hINF alpha 2b obtained with the PVX-based expression cassette was about 80 times higher than the yield of hINF alpha 2b obtained with a simple expression cassette in which the infα2b gene was controlled by the 35S promoter of cauliflower mosaic virus. KEY POINTS: ⢠PVX-based expression vectors provide efficient transient expression of infα2b gene ⢠N. benthamiana plants can produce human interferon alpha 2b at high levels ⢠The yield of the hINF α2b reached up to 1.2 mg/g of fresh leaf weight.
Subject(s)
Genetic Vectors , Interferon-alpha , Humans , Plants, Genetically Modified/genetics , Plants, Genetically Modified/metabolism , Interferon-alpha/genetics , Interferon-alpha/metabolism , Nicotiana/genetics , Promoter Regions, GeneticABSTRACT
Renal tumors comprise ~7% of all malignant pediatric tumors. Approximately 90% of pediatric kidney tumors comprise Wilms tumors, and the remaining 10% include clear cell sarcoma of the kidney, malignant rhabdoid tumor of the kidney, renal cell carcinoma and other rare renal tumors. Over the last 30 years, the role of cytokines and their receptors has been considerably investigated in both cancer progression and anti-cancer therapy. However, more effective immunotherapies require the cytokine profiling of each tumor type and comprehensive understanding of tumor biology. In this study, we aimed to investigate the activation of signaling pathways in response to cytokines in three pediatric kidney tumor cell lines, in WT-CLS1 and WT-3ab cells (both are Wilms tumors), and in G-401 cells (a rhabdoid kidney tumor, formerly classified as Wilms tumor). We observed that interferon-alpha (IFN-α) and interferon-gamma (IFN-γ) very strongly induced the activation of the STAT1 protein, whereas IL-6 and IFN-α activated STAT3 and IL-4 activated STAT6 in all examined tumor cell lines. STAT protein activation was examined by flow cytometry and Western blot using phospho-specific anti-STAT antibodies which recognize only activated (phosphorylated) STAT proteins. Nuclear translocation of phospho-STAT proteins upon activation with specific cytokines was furthermore confirmed by immunofluorescence. Our results also showed that both IFN-α and IFN-γ caused upregulation of major histocompatibility complex (MHC) class I proteins, however, these cytokines did not have any effect on the expression of MHC class II proteins. We also observed that pediatric kidney tumor cell lines exhibit the functional expression of an additional cytokine signaling pathway, the tumor necrosis factor (TNF)-α-mediated activation of nuclear factor kappa B (NF-κB). In summary, our data show that human pediatric renal tumor cell lines are responsive to stimulation with various human cytokines and could be used as in vitro models for profiling cytokine signaling pathways.
Subject(s)
Kidney Neoplasms , Tumor Necrosis Factor-alpha , Child , Humans , Tumor Necrosis Factor-alpha/metabolism , Cytokines/metabolism , Kidney Neoplasms/pathology , Interferon-alpha/metabolism , Histocompatibility Antigens Class I/metabolism , HLA Antigens , Cell Line, Tumor , STAT1 Transcription Factor/metabolism , Kidney/metabolismABSTRACT
A short 19 bp dsRNA with 3'-trinucleotide overhangs acting as immunostimulating RNA (isRNA) demonstrated strong antiproliferative action against cancer cells, immunostimulatory activity through activation of cytokines and Type-I IFN secretion, as well as anti-tumor and anti-metastatic effects in vivo. The aim of this study was to determine the tolerance of chemical modifications (2'-F, 2'-OMe, PS, cholesterol, and amino acids) located at different positions within this isRNA to its ability to activate the innate immune system. The obtained duplexes were tested in vivo for their ability to activate the synthesis of interferon-α in mice, and in tumor cell cultures for their ability to inhibit their proliferation. The obtained data show that chemical modifications in the composition of isRNA have different effects on its individual functions, including interferon-inducing and antiproliferative effects. The effect of modifications depends not only on the type of modification but also on its location and the surrounding context of the modifications. This study made it possible to identify leader patterns of modifications that enhance the properties of isRNA: F2/F2 and F2_S/F2 for interferon-inducing activity, as well as F2_S5/F2_S5, F2-NH2/F2-NH2, and Ch-F2/Ch-F2 for antiproliferative action. These modifications can improve the pharmacokinetic and pharmacodynamic properties, as well as increase the specificity of isRNA action to obtain the desired effect.
Subject(s)
Cell Proliferation , RNA, Double-Stranded , RNA, Double-Stranded/pharmacology , RNA, Double-Stranded/chemistry , Animals , Cell Proliferation/drug effects , Mice , Humans , Cell Line, Tumor , Interferon-alpha/metabolism , Adjuvants, Immunologic/pharmacology , Adjuvants, Immunologic/chemistry , Interferons/metabolismABSTRACT
The removal of double-stranded RNA (dsRNA) contaminants during in vitro mRNA synthesis is one of the technological problems to be solved. Apparently, these contaminants are the result of the T7 RNA polymerase side activity. In this study, we used a modified method of mRNA purification based on the selective binding of dsRNA to cellulose in ethanol-containing buffer. It was shown both in vivo and in vitro that the cellulose-purified mRNA preparation leads neither to activation of the lymphocyte inflammatory marker CD69 nor to increased release of IFNα in mice, and does not contain impurities detectable by antibodies to dsRNA.
Subject(s)
RNA, Double-Stranded , RNA, Messenger , Animals , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Double-Stranded/genetics , RNA, Double-Stranded/metabolism , Mice , Antigens, Differentiation, T-Lymphocyte/metabolism , Antigens, Differentiation, T-Lymphocyte/genetics , Antigens, CD/genetics , Antigens, CD/metabolism , Lectins, C-Type/metabolism , Lectins, C-Type/genetics , DNA-Directed RNA Polymerases/metabolism , DNA-Directed RNA Polymerases/genetics , Interferon-alpha/genetics , Interferon-alpha/metabolism , Interferon-alpha/biosynthesis , Viral Proteins/metabolism , Viral Proteins/geneticsABSTRACT
OBJECTIVE: To investigate the regulatory effect of interferon-α (IFN-α) on the apoptosis and killing function of CD56dimCD57+ natural killer (NK) cells in systemic lupus erythematosus (SLE) patients, and to explore the specific mechanism. METHODS: A total of sixty-four newly treated SLE patients and sixteen healthy controls (HC) enrolled in the Second Hospital of Dalian Medical University were selected as the research subjects. And the gene expression levels of molecules related to NK cell-killing function were detected by real-time quantitative polymerase chain reaction. CD56dimCD57+ NK cells were co-cultured with the K562 cells, and the apoptotic K562 cells were labeled with Annexin-â ¤ and 7-amino-actinomycin D. Peripheral blood mononuclear cells were treated with 20, 40, and 80 µmol/L hydrogen peroxide (H2O2), and treated without H2O2 as control, the expression level of perforin (PRF) was detected by flow cytometry. The concentration of IFN-α in serum was determined by enzyme linked immunosorbent assay. The expression levels of IFN-α receptors (IFNAR) on the surface of CD56dimCD57+ NK cells were detected by flow cytometry, and were represented by mean fluorescence intensity (MFI). CD56dimCD57+ NK cells were treated with 1 000 U/mL IFN-α for 24, 48 and 72 h, and no IFN-α treatment was used as the control, the apoptosis and the expression levels of mitochondrial reactive oxygen species (mtROS) were measured by flow cytometry and represented by MFI. RESULTS: Compared with HC(n=3), the expression levels of PRF1 gene in peripheral blood NK cells of the SLE patients (n=3) were decreased (1.24±0.41 vs. 0.57±0.12, P=0.05). Compared with HC(n=5), the ability of peripheral blood CD56dimCD57+ NK cells in the SLE patients (n=5) to kill K562 cells was significantly decreased (58.61%±10.60% vs. 36.74%±6.27%, P < 0.01). Compared with the control (n=5, 97.51%±1.67%), different concentrations of H2O2 treatment significantly down-regulated the PRF expression levels of CD56dimCD57+ NK cells in a dose-dependent manner, the 20 µmol/L H2O2 PRF was 83.23%±8.48% (n=5, P < 0.05), the 40 µmol/L H2O2 PRF was 79.53%±8.56% (n=5, P < 0.01), the 80 µmol/L H2O2 PRF was 76.67%±7.16% (n=5, P < 0.01). Compared to HC (n=16), the serum IFN-α levels were significantly increased in the SLE patients (n=45) with moderate to high systemic lupus erythematosus disease activity index (SLEDAI≥10) [(55.07±50.36) ng/L vs. (328.2±276.3) ng/L, P < 0.001]. Meanwhile, compared with HC (n=6), IFNAR1 expression in peripheral blood CD56dimCD57+ NK cells of the SLE patients (n=6) were increased (MFI: 292.7±91.9 vs. 483.2±160.3, P < 0.05), and compared with HC (n=6), IFNAR2 expression in peripheral blood CD56dimCD57+ NK cells of the SLE patients (n=7) were increased (MFI: 643.5±113.7 vs. 919.0±246.9, P < 0.05). Compared with control (n=6), the stimulation of IFN-α (n=6) significantly promoted the apoptosis of CD56dimCD57+ NK cells (20.48%±7.01% vs. 37.82%±5.84%, P < 0.05). In addition, compared with the control (n=4, MFI: 1 049±174.5), stimulation of CD56dimCD57+ NK cells with IFN-α at different times significantly promoted the production of mtROS in a time-dependent manner, 48 h MFI was 3 437±1 472 (n=4, P < 0.05), 72 h MFI was 6 495±1 089 (n=4, P < 0.000 1), but there was no significant difference at 24 h of stimulation. CONCLUSION: High serum IFN-α level in SLE patients may induce apoptosis by promoting mtROS production and inhibit perforin expression, which can down-regulate CD56dimCD57+ NK killing function.
Subject(s)
Interferon-alpha , Lupus Erythematosus, Systemic , Humans , Interferon-alpha/pharmacology , Interferon-alpha/metabolism , Perforin/metabolism , Leukocytes, Mononuclear/metabolism , Hydrogen Peroxide/metabolism , Interferon-gamma/metabolism , CD56 Antigen/metabolism , Killer Cells, Natural/metabolismABSTRACT
Synechocystis sp. PCC 6803 (Synechocystis) is a unicellular photosynthetic microorganism that has been used as a model for photo-biochemical research. It comprises a potential cell factory for the generation of valuable bioactive compounds, therapeutic proteins, and possibly biofuels. Fusion constructs of recombinant proteins with the CpcA α-subunit or CpcB ß-subunit of phycocyanin in Synechocystis have enabled true over-expression of several isoprenoid pathway enzymes and biopharmaceutical proteins to levels of 10-20 % of the total cellular protein. The present work employed the human interferon α-2 protein, as a study case of over-expression and downstream processing. It advanced the state of the art in the fusion constructs for protein overexpression technology by developing the bioresource for target protein separation from the fusion construct and isolation in substantially enriched or pure form. The work brings the cyanobacterial cell factory concept closer to meaningful commercial application for the photosynthetic production of useful recombinant proteins.
Subject(s)
Recombinant Proteins , Synechocystis , Synechocystis/metabolism , Humans , Recombinant Proteins/metabolism , Interferon-alpha/metabolism , Interferon alpha-2 , Protein BiosynthesisABSTRACT
Enhanced interferon α (IFNα) production has been implicated in the pathogenesis of systemic lupus erythematosus (SLE). We previously reported IFNα production by monocytes upon activation of the stimulator of IFN genes (STING) pathway was enhanced in patients with SLE. We investigated the mechanism of enhanced IFNα production in SLE monocytes. Monocytes enriched from the peripheral blood of SLE patients and healthy controls (HC) were stimulated with 2'3'-cyclic GAMP (2'3'-cGAMP), a ligand of STING. IFNα positive/negative cells were FACS-sorted for RNA-sequencing analysis. Gene expression in untreated and 2'3'-cGAMP-stimulated SLE and HC monocytes was quantified by real-time PCR. The effect of GATA binding protein 4 (GATA4) on IFNα production was investigated by overexpressing GATA4 in monocytic U937 cells by vector transfection. Chromatin immunoprecipitation was performed to identify GATA4 binding target genes in U937 cells stimulated with 2'3'-cGAMP. Differentially expressed gene analysis of cGAS-STING stimulated SLE and HC monocytes revealed the enrichment of gene sets related to cellular senescence in SLE. CDKN2A, a marker gene of cellular senescence, was upregulated in SLE monocytes at steady state, and its expression was further enhanced upon STING stimulation. GATA4 expression was upregulated in IFNα-positive SLE monocytes. Overexpression of GATA4 enhanced IFNα production in U937 cells. GATA4 bound to the enhancer region of IFIT family genes and promoted the expressions of IFIT1, IFIT2, and IFIT3, which promote type I IFN induction. SLE monocytes with accelerated cellular senescence produced high levels of IFNα related to GATA4 expression upon activation of the cGAS-STING pathway.
Subject(s)
GATA4 Transcription Factor , Gene Expression , Interferon-alpha , Lupus Erythematosus, Systemic , Humans , GATA4 Transcription Factor/genetics , GATA4 Transcription Factor/metabolism , Interferon Type I/metabolism , Interferon-alpha/metabolism , Lupus Erythematosus, Systemic/metabolism , Monocytes/metabolismABSTRACT
Toll-like receptor 9 (TLR-9) is a protein that helps our immune system identify specific DNA types. Upon detection, CpG oligodeoxynucleotides signal the immune system to generate cytokines, essential proteins that contribute to the body's defence against infectious diseases. Native phosphodiester type B CpG ODNs induce only Interleukin-6 with no effect on interferon-α. We prepared silicon quantum dots containing different surface charges, such as positive, negative, and neutral, using amine, acrylate-modified Plouronic F-127, and Plouronic F-127. Then, class B CpG ODNs are loaded on the surface of the prepared SiQDs. The uptake of ODNs varies based on the surface charge; positively charged SiQDs demonstrate higher adsorption compared to SiQDs with negative and neutral surface charges. The level of cytokine production in peripheral blood mononuclear cells was found to be associated with the surface charge of SiQDs prior to the binding of the CpG ODNs. Significantly higher levels of IL-6 and IFN-α induction were observed compared to neutral and negatively charged SiQDs loaded with CpG ODNs. This observation strongly supports the notion that the surface charge of SiQDs effectively regulates cytokine induction.
Subject(s)
Cytokines , Quantum Dots , Silicon , Quantum Dots/chemistry , Silicon/chemistry , Humans , Cytokines/metabolism , Leukocytes, Mononuclear/metabolism , Leukocytes, Mononuclear/immunology , Oligodeoxyribonucleotides/chemistry , Interleukin-6/metabolism , Surface Properties , Interferon-alpha/metabolism , Interferon-alpha/chemistry , Toll-Like Receptor 9/metabolismABSTRACT
Immunotherapy is widely used in cancer treatment; however, only a subset of patients responds well to it. Significant efforts have been made to identify patients who will benefit from immunotherapy. Successful anti-tumor immunity depends on an intact cancer-immunity cycle, especially long-lasting CD8+ T-cell responses. Interferon (IFN)-α/ß/IFN-γ/interleukin (IL)-15 pathways have been reported to be involved in the development of CD8+ T cells. And these pathways may predict responses to immunotherapy. Herein, we aimed to analyze multiple public databases to investigate whether IFN-α/ß/IFN-γ/IL-15 pathways could be used to predict the response to immunotherapy. Results showed that IFN-α/ß/IFN-γ/IL-15 pathways could efficiently predict immunotherapy response, and guanylate-binding protein 1 (GBP1) could represent the IFN-α/ß/IFN-γ/IL-15 pathways. In public and private cohorts, we further demonstrated that GBP1 could efficiently predict the response to immunotherapy. Functionally, GBP1 was mainly expressed in macrophages and strongly correlated with chemokines involved in T-cell migration. Therefore, our study comprehensively investigated the potential role of GBP1 in immunotherapy, which could serve as a novel biomarker for immunotherapy and a target for drug development.