ABSTRACT
Human T cells that express a T cell antigen receptor (TCR) containing γ-chain variable region 9 and δ-chain variable region 2 (Vγ9Vδ2) recognize phosphorylated prenyl metabolites as antigens in the presence of antigen-presenting cells but independently of major histocompatibility complex (MHC), the MHC class I-related molecule MR1 and antigen-presenting CD1 molecules. Here we used genetic approaches to identify the molecule that binds and presents phosphorylated antigens. We found that the butyrophilin BTN3A1 bound phosphorylated antigens with low affinity, at a stoichiometry of 1:1, and stimulated mouse T cells with transgenic expression of a human Vγ9Vδ2 TCR. The structures of the BTN3A1 distal domain in complex with host- or microbe-derived phosphorylated antigens had an immunoglobulin-like fold in which the antigens bound in a shallow pocket. Soluble Vγ9Vδ2 TCR interacted specifically with BTN3A1-antigen complexes. Accordingly, BTN3A1 represents an antigen-presenting molecule required for the activation of Vγ9Vδ2 T cells.
Subject(s)
Antigens, CD/metabolism , Antigens/immunology , Antigens/metabolism , Receptors, Antigen, T-Cell, gamma-delta/metabolism , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , Animals , Antigen Presentation/genetics , Antigen Presentation/immunology , Antigen-Presenting Cells/immunology , Antigen-Presenting Cells/metabolism , Antigens, CD/chemistry , Antigens, CD/genetics , Butyrophilins , Chromosomes, Human, Pair 6 , Humans , Mice , Mice, Transgenic , Models, Molecular , Organophosphates/chemistry , Organophosphates/metabolism , Phosphorylation , Protein Binding , Protein Conformation , Receptors, Antigen, T-Cell, gamma-delta/immunologyABSTRACT
BACKGROUND: Recognition of viral nucleic acids is one of the primary triggers for a type I interferon-mediated antiviral immune response. Inborn errors of type I interferon immunity can be associated with increased inflammation and/or increased susceptibility to viral infections as a result of dysbalanced interferon production. NFX1-type zinc finger-containing 1 (ZNFX1) is an interferon-stimulated double-stranded RNA sensor that restricts the replication of RNA viruses in mice. The role of ZNFX1 in the human immune response is not known. OBJECTIVE: We studied 15 patients from 8 families with an autosomal recessive immunodeficiency characterized by severe infections by both RNA and DNA viruses and virally triggered inflammatory episodes with hemophagocytic lymphohistiocytosis-like disease, early-onset seizures, and renal and lung disease. METHODS: Whole exome sequencing was performed on 13 patients from 8 families. We investigated the transcriptome, posttranscriptional regulation of interferon-stimulated genes (ISGs) and predisposition to viral infections in primary cells from patients and controls stimulated with synthetic double-stranded nucleic acids. RESULTS: Deleterious homozygous and compound heterozygous ZNFX1 variants were identified in all 13 patients. Stimulation of patient-derived primary cells with synthetic double-stranded nucleic acids was associated with a deregulated pattern of expression of ISGs and alterations in the half-life of the mRNA of ISGs and also associated with poorer clearance of viral infections by monocytes. CONCLUSION: ZNFX1 is an important regulator of the response to double-stranded nucleic acids stimuli following viral infections. ZNFX1 deficiency predisposes to severe viral infections and a multisystem inflammatory disease.
Subject(s)
Antigens, Neoplasm/genetics , Exome Sequencing , Genetic Predisposition to Disease , Primary Immunodeficiency Diseases/immunology , Virus Diseases/genetics , Antigens, Neoplasm/immunology , Child , Child, Preschool , Female , Humans , Infant , Inflammation/diagnostic imaging , Inflammation/genetics , Inflammation/immunology , Male , Primary Immunodeficiency Diseases/diagnostic imaging , Primary Immunodeficiency Diseases/genetics , Virus Diseases/diagnostic imaging , Virus Diseases/immunologyABSTRACT
To warrant the quality of the secretory proteome, stringent control systems operate at the endoplasmic reticulum (ER)-Golgi interface, preventing the release of nonnative products. Incompletely assembled oligomeric proteins that are deemed correctly folded must rely on additional quality control mechanisms dedicated to proper assembly. Here we unveil how ERp44 cycles between cisGolgi and ER in a pH-regulated manner, patrolling assembly of disulfide-linked oligomers such as IgM and adiponectin. At neutral, ER-equivalent pH, the ERp44 carboxy-terminal tail occludes the substrate-binding site. At the lower pH of the cisGolgi, conformational rearrangements of this peptide, likely involving protonation of ERp44's active cysteine, simultaneously unmask the substrate binding site and -RDEL motif, allowing capture of orphan secretory protein subunits and ER retrieval via KDEL receptors. The ERp44 assembly control cycle couples secretion fidelity and efficiency downstream of the calnexin/calreticulin and BiP-dependent quality control cycles.
Subject(s)
Membrane Proteins/metabolism , Molecular Chaperones/metabolism , Protein Multimerization , Amino Acid Motifs , Amino Acid Substitution , Catalytic Domain , Cell Cycle , Endoplasmic Reticulum/metabolism , Golgi Apparatus/metabolism , HeLa Cells , Humans , Hydrogen-Ion Concentration , Membrane Glycoproteins/metabolism , Membrane Proteins/chemistry , Membrane Proteins/genetics , Models, Molecular , Molecular Chaperones/chemistry , Molecular Chaperones/genetics , Mutagenesis, Site-Directed , Oxidoreductases/metabolism , Protein Transport , Secretory PathwayABSTRACT
BACKGROUND: Mutations in tetratricopeptide repeat domain 7A (TTC7A) and its mouse orthologue, Ttc7, result in a multisystemic disease, mostly affecting the epithelial barriers and immune system. Despite successful hematopoietic stem cell transplantation, ongoing progression of gastrointestinal manifestations can be life-threatening in TTC7A-deficient patients. OBJECTIVE: We sought to identify whether TTC7A mutations dysregulate epithelial cells only or whether a cell-intrinsic defect in lymphocytes or other cells contributes to disease manifestations. METHODS: Ttc7-mutated (Ttc7fsn/fsn) mice were crossed to generate double-mutant (Rag2-/-Ttc7fsn/fsn) and triple-mutant (Rag2-/-IL2rg-/-Ttc7fsn/fsn) mice. These models, together with bone marrow chimeras, were used to explore the role of adaptive and innate lymphocytes in the flaky skin phenotype. The effect of the Ttc7fsn/fsn mutation on stromal cells was tested in a xenograft model in conjunction with transcriptomic analysis of Ttc7fsn/fsn fibroblasts. RESULTS: We observed that the severity of epithelial hyperproliferation was accentuated by lymphocytes, whereas the phenotype was not induced by transfer of Ttc7-mutated hematopoietic cells. Furthermore, mice completely lacking the lymphocytic compartment were not protected from epithelial hyperproliferation. Ttc7-mutated mouse fibroblasts expressed increased transcript levels of insulin-like growth factor 1 (Igf1) and the antimicrobial protein regenerating islet-derived protein 3γ (Reg3γ). In a xenograft model Ttc7-mutated fibroblasts markedly increased epithelial proliferation of keratinocytes. Thus Ttc7-mutated fibroblasts were identified as potent instigators of epithelial hyperproliferation. CONCLUSION: Our results reveal a previously unsuspected fundamental cell-extrinsic role of Ttc7. We have identified potential candidates for molecularly targeted treatment strategies that will need to be evaluated in future preclinical studies.
Subject(s)
Cell Proliferation , Dermatitis/immunology , Epithelial Cells/immunology , Fibroblasts/immunology , Genetic Diseases, Inborn/immunology , Lymphocytes/immunology , Mutation , Proteins/immunology , Animals , BALB 3T3 Cells , Dermatitis/genetics , Dermatitis/pathology , Epithelial Cells/pathology , Fibroblasts/pathology , Genetic Diseases, Inborn/genetics , Genetic Diseases, Inborn/pathology , Humans , Lymphocytes/pathology , Mice , Mice, Knockout , Proteins/geneticsABSTRACT
The five-membered PRS gene family of Saccharomyces cerevisiae is an example of gene duplication allowing the acquisition of novel functions. Each of the five Prs polypeptides is theoretically capable of synthesising PRPP but at least one of the following heterodimers is required for survival: Prs1/Prs3, Prs2/Prs5 and Prs4/Prs5. Prs3 contains a pentameric motif 284KKCPK288 found only in nuclear proteins. Deletion of 284KKCPK288 destabilises the Prs1/Prs3 complex resulting in a cascade of events, including reduction in PRPP synthetase activity and altered cell wall integrity (CWI) as measured by caffeine sensitivity and Rlm1 expression. Prs3 also interacts with the kinetochore-associated protein, Nuf2. Following the possibility of 284KKCPK288-mediated transport of the Prs1/Prs3 complex to the nucleus, it may interact with Nuf2 and phosphorylated Slt2 permitting activation of Rlm1. This scenario explains the breakdown of CWI encountered in mutants lacking PRS3 or deleted for 284KKCPK288. However, removal of NHR1-1 from Prs1 does not disrupt the Prs1/Prs3 interaction as shown by increased PRPP synthetase activity. This is evidence for the separation of the two metabolic functions of the PRPP-synthesising machinery: provision of PRPP and maintenance of CWI and is an example of evolutionary development when multiple copies of a gene were present in the ancestral organism.
Subject(s)
Amino Acid Motifs , Microbial Viability , Phosphoribosyl Pyrophosphate/biosynthesis , Protein Subunits/metabolism , Ribose-Phosphate Pyrophosphokinase/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/physiology , Protein Binding , Protein Interaction Maps , Protein Subunits/genetics , Ribose-Phosphate Pyrophosphokinase/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Sequence DeletionABSTRACT
Psychosocial stress is a major risk factor for mood and anxiety disorders, in which excessive reactivity to aversive events/stimuli is a major psychopathology. In terms of pathophysiology, immune-inflammation is an important candidate, including high blood and brain levels of metabolites belonging to the kynurenine pathway. Animal models are needed to study causality between psychosocial stress, immune-inflammation and hyper-reactivity to aversive stimuli. The present mouse study investigated effects of psychosocial stress as chronic social defeat (CSD) versus control-handling (CON) on: Pavlovian tone-shock fear conditioning, activation of the kynurenine pathway, and efficacy of a specific inhibitor (IDOInh) of the tryptophan-kynurenine catabolising enzyme indoleamine 2,3-dioxygenase (IDO1), in reversing CSD effects on the kynurenine pathway and fear. CSD led to excessive fear learning and memory, whilst repeated oral escitalopram (antidepressant and anxiolytic) reversed excessive fear memory, indicating predictive validity of the model. CSD led to higher blood levels of TNF-α, IFN-γ, kynurenine (KYN), 3-hydroxykynurenine (3-HK) and kynurenic acid, and higher KYN and 3-HK in amygdala and hippocampus. CSD was without effect on IDO1 gene or protein expression in spleen, ileum and liver, whilst increasing liver TDO2 gene expression. Nonetheless, oral IDOInh reduced blood and brain levels of KYN and 3-HK in CSD mice to CON levels, and we therefore infer that CSD increases IDO1 activity by increasing its post-translational activation. Furthermore, repeated oral IDOInh reversed excessive fear memory in CSD mice to CON levels. IDOInh reversal of CSD-induced hyper-activity in the kynurenine pathway and fear system contributes significantly to the evidence for a causal pathway between psychosocial stress, immune-inflammation and the excessive fearfulness that is a major psychopathology in stress-related neuropsychiatric disorders.
Subject(s)
Brain/metabolism , Citalopram/pharmacology , Indoleamine-Pyrrole 2,3,-Dioxygenase/antagonists & inhibitors , Kynurenine/metabolism , Stress, Psychological/drug therapy , Stress, Psychological/metabolism , Animals , Antidepressive Agents, Second-Generation/pharmacology , Behavior, Animal/drug effects , Brain/drug effects , Brain/enzymology , Fear/drug effects , Indoleamine-Pyrrole 2,3,-Dioxygenase/metabolism , Kynurenic Acid/metabolism , Kynurenine/analogs & derivatives , Kynurenine/blood , Male , Mice , Mice, Inbred C57BL , Motor Activity/drug effects , Stress, Psychological/enzymology , Stress, Psychological/psychology , Tryptophan/metabolism , Tumor Necrosis Factor-alpha/metabolismSubject(s)
Anemia, Hemolytic, Autoimmune/genetics , Asthma/genetics , Erythrocytes/physiology , Germ-Line Mutation/genetics , STAT3 Transcription Factor/genetics , Anemia, Hemolytic, Autoimmune/drug therapy , Cell Differentiation , Cell Proliferation , Cells, Cultured , Child , Erythropoiesis/genetics , Erythropoietin/metabolism , Gene Expression Regulation , Humans , Iron Chelating Agents/therapeutic use , Receptors, Erythropoietin/metabolism , STAT5 Transcription Factor/metabolism , Severity of Illness Index , Signal Transduction , Exome Sequencing , beta-Globins/genetics , beta-Globins/isolation & purificationSubject(s)
CD48 Antigen/genetics , CD8-Positive T-Lymphocytes/immunology , Cytotoxicity, Immunologic/genetics , Interleukin-6/metabolism , Killer Cells, Natural/immunology , Lymphohistiocytosis, Hemophagocytic/genetics , Mutation/genetics , Computational Biology , HEK293 Cells , Heterozygote , Humans , Inflammation , Interleukin-6/genetics , Pedigree , Qa-SNARE Proteins/genetics , Recurrence , Up-Regulation , Exome Sequencing , Young AdultABSTRACT
In the early secretory compartment (ESC), a network of chaperones and enzymes assists oxidative folding of nascent proteins. Ero1 flavoproteins oxidize protein disulfide isomerase (PDI), generating H2O2 as a byproduct. Peroxiredoxin 4 (Prx4) can utilize luminal H2O2 to oxidize PDI, thus favoring oxidative folding while limiting oxidative stress. Interestingly, neither ER oxidase contains known ER retention signal(s), raising the question of how cells prevent their secretion. Here we show that the two proteins share similar intracellular localization mechanisms. Their secretion is prevented by sequential interactions with PDI and ERp44, two resident proteins of the ESC-bearing KDEL-like motifs. PDI binds preferentially Ero1α, whereas ERp44 equally retains Ero1α and Prx4. The different binding properties of Ero1α and Prx4 increase the robustness of ER redox homeostasis.
Subject(s)
Membrane Glycoproteins/metabolism , Oxidoreductases/metabolism , Peroxiredoxins/metabolism , Secretory Pathway , Amino Acid Sequence , Blotting, Western , Endoplasmic Reticulum/metabolism , HEK293 Cells , HeLa Cells , Homeostasis , Humans , Kinetics , Membrane Glycoproteins/genetics , Membrane Proteins/genetics , Membrane Proteins/metabolism , Microscopy, Confocal , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Molecular Sequence Data , Mutation , Oxidation-Reduction , Oxidoreductases/genetics , Peroxiredoxins/genetics , Protein Binding , Protein Disulfide-Isomerases/genetics , Protein Disulfide-Isomerases/metabolism , RNA Interference , Surface Plasmon ResonanceABSTRACT
In the endoplasmic reticulum (ER) of eukaryotic cells, Ero1 flavoenzymes promote oxidative protein folding through protein disulphide isomerase (PDI), generating reactive oxygen species (hydrogen peroxide) as byproducts. Therefore, Ero1 activity must be strictly regulated to avoid futile oxidation cycles in the ER. Although regulatory mechanisms restraining Ero1α activity ensure that not all PDIs are oxidized, its specificity towards PDI could allow other resident oxidoreductases to remain reduced and competent to carry out isomerization and reduction of protein substrates. In this study, crystal structures of human Ero1α were solved in its hyperactive and inactive forms. Our findings reveal that human Ero1α modulates its oxidative activity by properly positioning regulatory cysteines within an intrinsically flexible loop, and by fine-tuning the electron shuttle ability of the loop through disulphide rearrangements. Specific PDI targeting is guaranteed by electrostatic and hydrophobic interactions of Ero1α with the PDI b'-domain through its substrate-binding pocket. These results reveal the molecular basis of the regulation and specificity of protein disulphide formation in human cells.
Subject(s)
Endoplasmic Reticulum/metabolism , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/metabolism , Models, Molecular , Oxidoreductases/chemistry , Oxidoreductases/metabolism , Protein Disulfide-Isomerases/metabolism , Crystallography, X-Ray , Humans , Hydrophobic and Hydrophilic Interactions , Oxidation-Reduction , Protein Binding , Static ElectricityABSTRACT
Introduction: Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening immune disorder characterized by uncontrolled lymphocyte and macrophage activation and a subsequent cytokine storm. The timely initiation of immunosuppressive treatment is crucial for survival. Methods: Here, we harnessed Vγ9Vδ2 T cell degranulation to develop a novel functional assay for the diagnosis of HLH. We compared the novel assay with the conventional natural killer (NK) cell stimulation method in terms of efficiency, specificity, and reliability. Our analysis involved 213 samples from 182 individuals, including 23 samples from 12 patients with degranulation deficiency (10 individuals with UNC13D deficiency, 1 with STXBP2 deficiency, and 1 with RAB27A deficiency). Results: While both tests exhibited 100% sensitivity, the Vγ9Vδ2 T cell degranulation assay showed a superior specificity of 86.2% (n=70) compared to the NK cell degranulation assay, which achieved 78.9% specificity (n=213). The Vγ9Vδ2 T cell degranulation assay offered simpler technical requirements and reduced labor intensity, leading to decreased susceptibility to errors with faster processing times. Discussion: This efficiency stemmed from the sole requirement of dissolving (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) powder, contrasting with the intricate maintenance of K562 cells necessary for the NK cell degranulation assay. With its diminished susceptibility to errors, we anticipate that the assay will require fewer repetitions of analysis, rendering it particularly well-suited for testing infants. Conclusion: The Vγ9Vδ2 T cell degranulation assay is a user-friendly, efficient diagnostic tool for HLH. It offers greater specificity, reliability, and practicality than established methods. We believe that our present findings will facilitate the prompt, accurate diagnosis of HLH and thus enable rapid treatment and better patient outcomes.
Subject(s)
Cell Degranulation , Killer Cells, Natural , Lymphohistiocytosis, Hemophagocytic , Humans , Lymphohistiocytosis, Hemophagocytic/diagnosis , Lymphohistiocytosis, Hemophagocytic/immunology , Lymphohistiocytosis, Hemophagocytic/genetics , Female , Male , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Receptors, Antigen, T-Cell, gamma-delta/metabolism , Receptors, Antigen, T-Cell, gamma-delta/genetics , Child, Preschool , Child , Infant , Adolescent , rab27 GTP-Binding Proteins/genetics , Membrane Proteins/genetics , Membrane Proteins/metabolism , Adult , T-Lymphocytes/immunology , Reproducibility of Results , Lymphocyte Activation , Sensitivity and Specificity , Munc18 ProteinsABSTRACT
CD40L (CD154) is regulated at the posttranscriptional level by an activation-induced process that results in a highly stable transcript at extended times of T cell activation. Transcript stability is mediated by polypyrimidine tract-binding protein (PTB)-containing complexes (complex I and II) that bind to three adjacent CU-rich sequences within the 3' untranslated region. To assess the role of PTB in the expression and distribution of CD40L mRNA, PTB was targeted using short hairpin RNA in both primary T cells and a T cell line that recapitulates the stability phase of regulated CD40L mRNA decay. PTB knockdown resulted in a marked decrease in the mRNA stability that resulted in lowered CD40L surface expression. PTB was also critical for appropriate distribution of CD40L mRNA between the nucleus and cytoplasm and in the cytoplasm between the cytosol and the translating polysomes. The activation-induced formation of PTB-specific ribonucleoprotein complexes was observed only with cytoplasmic and not nuclear PTB indicating functional differences in the protein defined by cellular localization. Finally, we observed that cytoplasmic and nuclear PTB isoforms were differentially modified relative to each other and that the changes in cytoplasmic PTB were consistent with activation-induced phosphorylation. Together this work suggests that differentially modified PTB regulates CD40L expression at multiple steps by 1) retaining CD40L mRNA in the nucleus, 2) directly regulating mRNA stability at late times of activation, and 3) forming a ribonuclear complex that preferentially associates with translating ribosomes thus leading to an enhanced level of CD40L protein.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , CD40 Ligand/genetics , CD40 Ligand/metabolism , Polypyrimidine Tract-Binding Protein/physiology , RNA, Messenger/metabolism , Cells, Cultured , Cytoplasm/genetics , Cytoplasm/metabolism , HEK293 Cells , Humans , Jurkat Cells , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Nuclear Proteins/physiology , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Isoforms/physiology , Protein Transport/genetics , Protein Transport/immunology , RNA Stability/genetics , RNA Stability/immunology , Subcellular Fractions/metabolism , Transcription, Genetic/immunologyABSTRACT
Cytotoxic lymphocytes kill target cells through polarized release of the content of cytotoxic granules towards the target cell. The importance of this cytotoxic pathway in immune regulation is evidenced by the severe and often fatal condition, known as hemophagocytic lymphohistiocytosis (HLH) that occurs in mice and humans with inborn errors of lymphocyte cytotoxic function. The clinical and preclinical data indicate that the damage seen in severe, virally triggered HLH is due to an overwhelming immune system reaction and not the direct effects of the virus per se. The main HLH-disease mechanism, which links impaired cytotoxicity to excessive release of pro-inflammatory cytokines is a prolongation of the synapse time between the cytotoxic effector cell and the target cell, which prompts the former to secrete larger amounts of cytokines (including interferon gamma) that activate macrophages. We and others have identified novel genetic HLH spectrum disorders. In the present update, we position these newly reported molecular causes, including CD48-haploinsufficiency and ZNFX1-deficiency, within the pathogenic pathways that lead to HLH. These genetic defects have consequences on the cellular level on a gradient model ranging from impaired lymphocyte cytotoxicity to intrinsic activation of macrophages and virally infected cells. Altogether, it is clear that target cells and macrophages may play an independent role and are not passive bystanders in the pathogenesis of HLH. Understanding these processes which lead to immune dysregulation may pave the way to novel ideas for medical intervention in HLH and virally triggered hypercytokinemia.
Subject(s)
Cytokines , Lymphohistiocytosis, Hemophagocytic , Humans , Animals , Mice , Cytokines/metabolism , Lymphohistiocytosis, Hemophagocytic/etiology , Lymphocytes/metabolism , Cytotoxicity, Immunologic , Interferon-gammaABSTRACT
In the endoplasmic reticulum (ER) of human cells, ERO1α and protein-disulfide isomerase (PDI) constitute one of the major electron flow pathways that catalyze oxidative folding of secretory proteins. Specific and limited PDI oxidation by ERO1α is essential to avoid ER hyperoxidation. To investigate how ERO1α oxidizes PDI selectively among more than 20 ER-resident PDI family member proteins, we performed docking simulations and systematic biochemical analyses. Our findings reveal that a protruding ß-hairpin of ERO1α specifically interacts with the hydrophobic pocket present in the redox-inactive PDI b'-domain through the stacks between their aromatic residues, leading to preferred oxidation of the C-terminal PDI a'-domain. ERO1α associated preferentially with reduced PDI, explaining the stepwise disulfide shuttle mechanism, first from ERO1α to PDI and then from oxidized PDI to an unfolded polypeptide bound to its hydrophobic pocket. The interaction of ERO1α with ERp44, another PDI family member protein, was also analyzed. Notably, ERO1α-dependent PDI oxidation was inhibited by a hyperactive ERp44 mutant that lacks the C-terminal tail concealing the substrate-binding hydrophobic regions. The potential ability of ERp44 to inhibit ERO1α activity may suggest its physiological role in ER redox and protein homeostasis.
Subject(s)
Disulfides/chemistry , Membrane Glycoproteins/chemistry , Oxidoreductases/chemistry , Protein Disulfide-Isomerases/chemistry , Protein Folding , Binding Sites , Catalysis , Disulfides/metabolism , Humans , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Membrane Proteins/chemistry , Membrane Proteins/genetics , Membrane Proteins/metabolism , Molecular Chaperones/chemistry , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Oxidation-Reduction , Oxidoreductases/genetics , Oxidoreductases/metabolism , Protein Disulfide-Isomerases/genetics , Protein Disulfide-Isomerases/metabolism , Protein Structure, SecondaryABSTRACT
Pathogen-associated molecular patterns, including cytoplasmic DNA and double-strand (ds)RNA trigger the induction of interferon (IFN) and antiviral states protecting cells and organisms from pathogens. Here we discovered that the transfection of human airway cell lines or non-transformed fibroblasts with 24mer dsRNA mimicking the cellular micro-RNA (miR)29b-1* gives strong anti-viral effects against human adenovirus type 5 (AdV-C5), influenza A virus X31 (H3N2), and SARS-CoV-2. These anti-viral effects required blunt-end complementary RNA strands and were not elicited by corresponding single-strand RNAs. dsRNA miR-29b-1* but not randomized miR-29b-1* mimics induced IFN-stimulated gene expression, and downregulated cell adhesion and cell cycle genes, as indicated by transcriptomics and IFN-I responsive Mx1-promoter activity assays. The inhibition of AdV-C5 infection with miR-29b-1* mimic depended on the IFN-alpha receptor 2 (IFNAR2) and the RNA-helicase retinoic acid-inducible gene I (RIG-I) but not cytoplasmic RNA sensors MDA5 and ZNFX1 or MyD88/TRIF adaptors. The antiviral effects of miR29b-1* were independent of a central AUAU-motif inducing dsRNA bending, as mimics with disrupted AUAU-motif were anti-viral in normal but not RIG-I knock-out (KO) or IFNAR2-KO cells. The screening of a library of scrambled short dsRNA sequences identified also anti-viral mimics functioning independently of RIG-I and IFNAR2, thus exemplifying the diverse anti-viral mechanisms of short blunt-end dsRNAs.
Subject(s)
COVID-19 , Interferon Type I , MicroRNAs , Antiviral Agents/pharmacology , DEAD Box Protein 58/genetics , DEAD Box Protein 58/metabolism , DEAD-box RNA Helicases/genetics , Humans , Influenza A Virus, H3N2 Subtype/genetics , Interferon Type I/genetics , RNA, Double-Stranded , SARS-CoV-2ABSTRACT
Thymic epithelial cells (TECs) are essential in supporting the development of mature T cells from hematopoietic progenitor cells and facilitate their lineage-commitment, proliferation, T-cell receptor repertoire selection and maturation. While animal model systems have greatly aided in elucidating the contribution of stromal cells to these intricate processes, human tissue has been more difficult to study, partly due to a lack of suitable surface markers comprehensively defining human TECs. Here, we conducted a flow cytometry based surface marker screen to reliably identify and quantify human TECs and delineate medullary from cortical subsets. These findings were validated by transcriptomic and histologic means. The combination of EpCAM, podoplanin (pdpn), CD49f and CD200 comprehensively identified human TECs and not only allowed their reliable distinction in medullary and cortical subsets but also their detailed quantitation. Transcriptomic profiling of each subset in comparison to fibroblasts and endothelial cells confirmed the identity of the different stromal cell subsets sorted according to the proposed strategy. Our dataset not only demonstrated transcriptional similarities between TEC and cells of mesenchymal origin but furthermore revealed a subset-specific distribution of a specific set of extracellular matrix-related genes in TECs. This indicates that TECs significantly contribute to the distinct compartmentalization - and thus function - of the human thymus. We applied the strategy to quantify TEC subsets in 31 immunologically healthy children, which revealed sex-specific differences of TEC composition early in life. As the distribution of mature CD4- or CD8-single-positive thymocytes was correspondingly altered, the composition of the thymic epithelial compartment may directly impact on the CD4-CD8-lineage choice of thymocytes. We prove that the plain, reliable strategy proposed here to comprehensively identify human TEC subpopulations by flow cytometry based on surface marker expression is suitable to determine their frequency and phenotype in health and disease and allows sorting of live cells for downstream analysis. Its use reaches from a reliable diagnostic tool for thymic biopsies to improved phenotypic characterization of thymic grafts intended for therapeutic use.
Subject(s)
Cell Separation , Epithelial Cells/metabolism , Flow Cytometry , Gene Expression Profiling , Stromal Cells/metabolism , Thymus Gland/metabolism , Transcriptome , 22q11 Deletion Syndrome/genetics , 22q11 Deletion Syndrome/immunology , 22q11 Deletion Syndrome/metabolism , Adolescent , Age Factors , Biomarkers/metabolism , Child , Child, Preschool , Chromosome Deletion , Chromosomes, Human, Pair 22 , Epithelial Cells/immunology , Female , Humans , Infant , Infant, Newborn , Male , Myasthenia Gravis/genetics , Myasthenia Gravis/immunology , Myasthenia Gravis/metabolism , Phenotype , Sex Factors , Stromal Cells/immunology , Thymus Gland/cytology , Thymus Gland/immunologyABSTRACT
BACKGROUND: Recently, cases of multisystem inflammatory syndrome in children (MIS-C) associated with coronavirus disease 2019 (COVID-19) have been reported worldwide. Negative polymerase chain reaction (RT-PCR) testing associated with positive serology in most of the cases suggests a postinfectious syndrome. Because the pathophysiology of this syndrome is still poorly understood, extensive virological and immunological investigations are needed. METHODS: We report a series of 4 pediatric patients admitted to Geneva University Hospitals with persistent fever and laboratory evidence of inflammation meeting the published definition of MIS-C related to COVID-19, to whom an extensive virological and immunological workup was performed. RESULTS: RT-PCRs on multiple anatomical compartments were negative, whereas anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunoglobulin A (IgA) and immunoglobulin G (IgG) were strongly positive by enzyme-linked immunosorbent assay and immunofluorescence. Both pseudoneutralization and full virus neutralization assays showed the presence of neutralizing antibodies in all children, confirming a recent infection with SARS-CoV-2. The analyses of cytokine profiles revealed an elevation in all cytokines, as reported in adults with severe COVID-19. Although differing in clinical presentation, some features of MIS-C show phenotypic overlap with hemophagocytic lymphohistiocytosis (HLH). In contrast to patients with primary HLH, our patients showed normal perforin expression and natural killer (NK) cell degranulation. The levels of soluble interleukin (IL)-2 receptor (sIL-2R) correlated with the severity of disease, reflecting recent T-cell activation. CONCLUSION: Our findings suggest that MIS-C related to COVID-19 is caused by a postinfectious inflammatory syndrome associated with an elevation in all cytokines, and markers of recent T-cell activation (sIL-2R) occurring despite a strong and specific humoral response to SARS-CoV-2. Further functional and genetic analyses are essential to better understand the mechanisms of host-pathogen interactions.
Subject(s)
COVID-19 , Antibodies, Neutralizing , Child , Humans , SARS-CoV-2 , Systemic Inflammatory Response SyndromeABSTRACT
Most children with a SARS-CoV-2 infection are asymptomatic or exhibit mild symptoms. However, a small number of children develop features of substantial inflammation temporarily related to the COVID-19 also called multisystem inflammatory syndrome in children (MIS-C) or pediatric inflammatory multisystem syndrome temporally associated with SARS-CoV-2 (PIMS-TS), clinically similar to Kawasaki disease, toxic shock syndrome and hemophagocytic lymphohistiocytosis (HLH). It is well-known that genetic pre-disposition plays an important role in virally-triggered diseases such as Epstein-Barr virus (EBV)-associated HLH, while this has not yet been established for patients with MIS-C. Here we describe a male patient fulfilling the diagnostic criteria of MIS-C, who was initially treated according to current consensus guidelines. Presence of hypofibrinogenemia, normal lymphocyte counts and C-reactive protein, but substantial hyperferritinemia distinguish this patient from others with MIS-C. The clinical course following initial presentation with acute respiratory distress syndrome was marked by fatal liver failure in the context of EBV-associated HLH despite treatment with steroids, intravenous immunoglobulins, interleukin (IL)-1 receptor blockade and eventually HLH-directed treatment. X-linked lymphoproliferative disease type 1 (XLP1), a subtype of primary HLH was diagnosed in this patient post-mortem. This case report highlights the importance of including HLH in the differential diagnosis in MIS-C with severe disease course to allow specific, risk-adapted treatment and genetic counseling.
ABSTRACT
Interactions between CD40 and its ligand CD154 are involved in the progression of both cell mediated and innate immunity. These interactions are brought about by the transient expression of CD154 on activated CD4(+) T cells, which is regulated, in part, at the level of mRNA turnover. Here we have focused on analyzing the pattern of post-transcriptional regulation in mouse CD4(+) T cells in response to activation. Initial experiments identify a region of the murine CD154 mRNA that binds a polypyrimidine tract-binding protein-containing complex (mComplex I), which is activation-dependent and binds to a single CU-rich site within the 3' uTR Subsequent findings demonstrate that in vivo polyclonal activation of T cells leads to a pattern of differential CD154 mRNA stability that is directly dependent on extent of activation. Furthermore, in vitro activation of antigen-primed T cells shows that the CD154 mRNA half-life increases relative to that of unprimed cells. Importantly, this is the first report demonstrating that the regulation of CD154 in vivo is connected to an activation-induced program of mRNA decay and thus provides strong evidence for post-transcriptional mechanisms having a physiological role in regulating CD154 expression during an ongoing immune response.
Subject(s)
CD4-Positive T-Lymphocytes/metabolism , CD40 Ligand/genetics , RNA Processing, Post-Transcriptional , RNA Stability , 3' Untranslated Regions/genetics , 3' Untranslated Regions/metabolism , Animals , Antibodies, Monoclonal/pharmacology , Antigens/immunology , Base Sequence , CD28 Antigens/immunology , CD4-Positive T-Lymphocytes/drug effects , CD4-Positive T-Lymphocytes/immunology , Electrophoretic Mobility Shift Assay , Flow Cytometry , Humans , Jurkat Cells , Lymphocyte Activation/drug effects , Lymphocyte Activation/immunology , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Polypyrimidine Tract-Binding Protein/metabolism , Protein Binding , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Nucleic Acid , Time FactorsABSTRACT
ERp44 mediates thiol-dependent retention in the early secretory pathway, forming mixed disulphides with substrate proteins through its conserved CRFS motif. Here, we present its crystal structure at a resolution of 2.6 A. Three thioredoxin domains-a, b and b'-are arranged in a clover-like structure. A flexible carboxy-terminal tail turns back to the b' and a domains, shielding a hydrophobic pocket in domain b' and a hydrophobic patch around the CRFS motif in domain a. Mutational and functional studies indicate that the C-terminal tail gates the CRFS area and the adjacent hydrophobic pocket, dynamically regulating protein quality control.