ABSTRACT
Peroxisomes have long been established to play a central role in regulating various metabolic activities in mammalian cells. These organelles act in concert with mitochondria to control the metabolism of lipids and reactive oxygen species. However, while mitochondria have emerged as an important site of antiviral signal transduction, a role for peroxisomes in immune defense is unknown. Here, we report that the RIG-I-like receptor (RLR) adaptor protein MAVS is located on peroxisomes and mitochondria. We find that peroxisomal and mitochondrial MAVS act sequentially to create an antiviral cellular state. Upon viral infection, peroxisomal MAVS induces the rapid interferon-independent expression of defense factors that provide short-term protection, whereas mitochondrial MAVS activates an interferon-dependent signaling pathway with delayed kinetics, which amplifies and stabilizes the antiviral response. The interferon regulatory factor IRF1 plays a crucial role in regulating MAVS-dependent signaling from peroxisomes. These results establish that peroxisomes are an important site of antiviral signal transduction.
Subject(s)
Immunity, Innate , Peroxisomes/metabolism , Signal Transduction , Adaptor Proteins, Signal Transducing/immunology , Adaptor Proteins, Signal Transducing/metabolism , Animals , Cell Line , Chlorocebus aethiops , Fibroblasts/metabolism , Hepatocytes/metabolism , Humans , Interferons/metabolism , Mice , Mitochondria/metabolism , Vero CellsABSTRACT
During the course of a viral infection, viral proteins interact with an array of host proteins and pathways. Here, we present a systematic strategy to elucidate the dynamic interactions between H1N1 influenza and its human host. A combination of yeast two-hybrid analysis and genome-wide expression profiling implicated hundreds of human factors in mediating viral-host interactions. These factors were then examined functionally through depletion analyses in primary lung cells. The resulting data point to potential roles for some unanticipated host and viral proteins in viral infection and the host response, including a network of RNA-binding proteins, components of WNT signaling, and viral polymerase subunits. This multilayered approach provides a comprehensive and unbiased physical and regulatory model of influenza-host interactions and demonstrates a general strategy for uncovering complex host-pathogen relationships.
Subject(s)
Host-Pathogen Interactions , Influenza A Virus, H1N1 Subtype/metabolism , Viral Proteins/metabolism , Apoptosis , Epithelial Cells/virology , Gene Expression Profiling , Humans , Influenza A Virus, H1N1 Subtype/immunology , Influenza A Virus, H1N1 Subtype/pathogenicity , Interferons/metabolism , Lung/cytology , Lung/virology , Proteomics , RNA, Small Interfering/metabolism , RNA, Viral/metabolism , Two-Hybrid System Techniques , Viral Nonstructural Proteins/metabolism , Wnt Proteins/metabolismABSTRACT
BACKGROUND: Newborn screening (NBS) is an effective public health intervention that reduces death and disability from treatable genetic diseases, but many conditions are not screened due to a lack of a suitable assay. Whole genome and whole exome sequencing can potentially expand NBS but there remain many technical challenges preventing their use in population NBS. We investigated if targeted gene sequencing (TGS) is a feasible methodology for expanding NBS. METHODS: We constructed a TGS panel of 164 genes which screens for a broad range of inherited conditions. We designed a high-volume, low-turnaround laboratory and bioinformatics workflow that avoids the technical and data interpretation challenges associated with whole genome and whole exome sequencing. A methods-based analytical validation of the assay was completed and test performance in 2552 newborns examined. We calculated annual birth estimates for each condition to assess cost-effectiveness. RESULTS: Assay analytical sensitivity was >99% and specificity was 100%. Of the newborns screened, 1.3% tested positive for a condition. On average, each individual had 225 variants to interpret and 1.8% were variants of uncertain significance (VUS). The turnaround time was 7 to 10 days. Maximum batch size was 1536 samples. CONCLUSIONS: We demonstrate that a TGS assay could be incorporated into an NBS program soon to increase the number of conditions screened. Additionally, we conclude that NBS using TGS may be cost-effective.
Subject(s)
Computational Biology , Neonatal Screening , Infant, Newborn , Humans , Neonatal Screening/methods , Feasibility Studies , DNA , Sequence Analysis, DNAABSTRACT
OBJECTIVE: European and Australian guidelines for cystic fibrosis (CF) reproductive carrier screening recommend testing a small number of high frequency CF causing variants, rather than comprehensive CFTR sequencing. The study objective was to determine variant detection rates of commercially available targeted reproductive carrier screening tests in Australia. METHODS: Next-generation DNA sequencing of the CFTR gene was performed on 2552 individuals from a whole population sample to identify CF causing variants. The variant detection rates of two commercially available Australian reproductive carrier screening tests, which target 50 or 175 CF causing variants, in this population were calculated. The ethnicity of individuals was determined using principal component analysis. RESULTS: Variant detection rates of the tests for 50 and 175 CF causing variants were 88.2% and 90.8%, respectively. No CF causing variants in individuals of East Asian ethnicity (n = 3) were detected by either test, while >86.6% (n = 69) of CF causing variants in Europeans would be identified by either test. CONCLUSIONS: Reproductive carrier screening tests for a targeted set of high frequency CF variants are unable to detect approximately 10% of CF variants in a multiethnic Australian population, and individuals of East Asian ethnicity are disproportionally affected by this test limitation.
Subject(s)
Cystic Fibrosis , Humans , Cystic Fibrosis/diagnosis , Cystic Fibrosis/epidemiology , Cystic Fibrosis/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Australia/epidemiology , Genetic Testing , Ethnicity , MutationABSTRACT
BACKGROUND: RNA interference (RNAi) provides a powerful way to investigate the role of genes in disease pathogenesis and modulate gene expression to treat disease. In 2018, the FDA approved patisiran, the first RNAi-based drug, hence paving the way for a novel class of RNAi therapeutics. Harnessing RNAi to inhibit vaginal HIV transmission requires effective gene silencing in immune cells, which remains difficult. Knockdown in accessible mucosal tissues may be easier than systemic gene silencing. Vaginally applied cholesterol-conjugated small interfering RNAs (chol-siRNAs) blocked herpes simplex virus transmission in mice without tissue damage or immunostimulation. OBJECTIVES AND METHODS: To investigate using flow cytometry, confocal microscopy, and quantitative imaging if chol-siRNAs silence gene expression in vaginal immune cells in mice. RESULTS: Although chol-siRNAs and lipoplexed-siRNAs silence gene expression in dendritic cells (DCs) in vitro, most internalized siRNAs concentrate within multivesicular bodies, where they are inaccessible to the cellular RNAi machinery. When applied intravaginally in vivo, chol-siRNAs penetrate the vaginal mucosa, including the lamina propria, and are efficiently internalized by intraepithelial (IE) and lamina propria (LP) DCs, and CD11b+ CD45+ cells, but not by T cells. Chol-siRNAs induce partial gene silencing in IE and LP DCs throughout the genital mucosa in vivo but are inactive in F4/80+ CD11b+ macrophages and T cells. CONCLUSION: As mucosal DCs play an essential role for mucosal viral entry and dissemination, chol-siRNAs could be harnessed to target various host factors that are critical for viral uptake, DC migration and trans-infection of virions to T cells, hence allowing the development of a preventive vaginal HIV microbicide. Furthermore, chol-siRNAs could help elucidate the pathways of HIV transmission and understand the immunologic function of DCs in the genital tract.
Subject(s)
HIV Infections , Female , Mice , Animals , RNA Interference , Dendritic Cells/metabolism , Mucous Membrane , Gene ExpressionABSTRACT
BACKGROUND: Cystic Fibrosis (CF) is one of the most prevalent autosomal recessive inherited disease in Caucasians. Rates of CF were thought to be negligible in non-Caucasians but growing epidemiological evidence shows CF is more common in Indian, African, Hispanic, Asian, and other ethnic groups than previously thought. Almost all second-tier molecular diagnostic tools currently used to confirm the diagnosis of CF consist of panels of the most common CF-causing DNA variants in Caucasians. However non-Caucasian individuals with CF often have a different spectrum of pathogenic variants than Caucasians, limiting the clinical utility of existing molecular diagnostic panels in this group. As a consequence of racially inequitable CF testing frameworks, non-Caucasians with CF encounter greater delays in diagnosis and associated harms than Caucasians. An unbiased approach of detecting CF-causing DNA variants using full gene sequencing could potentially address racial inequality in current CF testing. CASE PRESENTATION: We present the case of a female baby from rural India who had a borderline first-tier newborn screening result for CF. Instead of choosing a targeted CF panel for second-tier testing, we used next-generation DNA sequencing to comprehensively analyze the cystic fibrosis transmembrane conductance regulator gene as an unbiased approach for molecular confirmation of CF. Sequencing identified two pathogenic variants that cause CF. One variant (c.1521_1523delCTT) is the most common cause of CF, while the other variant (c.870-1G > C) is absent from all population allele databases and has not been found in the Indian population previously. The rare variant would not have been detected by all currently available targeted CF panels used for second- or third-tier molecular CF testing. CONCLUSIONS: Our use of full gene sequencing as a second-tier CF test in a non-Caucasian patient avoided the problems of missed diagnosis from using Caucasian-biased targeted CF panels currently recommended for second-tier testing. Full gene sequencing should be considered as the standard methodology of second-tier CF testing to enable equal opportunity for CF diagnosis in non-Caucasians.
Subject(s)
Cystic Fibrosis , Cystic Fibrosis/diagnosis , Cystic Fibrosis/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , DNA , Female , Humans , India , Infant, Newborn , Mutation , Sequence Analysis, DNAABSTRACT
Newborn screening (NBS) assays for spinal muscular atrophy (SMA) typically use a polymerase chain reaction (PCR) based assay to identify individuals with homozygous deletion in exon 7 of the SMN1 gene. Due to high DNA sequence homology between SMN1 and SMN2, it has previously been difficult to accurately bioinformatically map short reads from next-generation DNA sequencing (NGS) to SMN1, resulting in low analytical performance and preventing NGS being used for SMA screening. Advances in bioinformatics have allowed NGS to be used in diagnostic settings, but to date these assays have not reached the scale required for high volume population newborn screening and have not been performed on the dried blood spot samples that NBS programs currently use. Here we integrate an NGS assay using hybridisation-based capture with a customised bioinformatics algorithm and purpose designed high throughput reporting software into an existing NBS program to achieve a laboratory workflow for population SMA screening. We tested the NGS assay on over 2500 newborns born over 2 weeks in a NBS program in a technical feasibility study and show high sensitivity and specificity. Our results suggest NGS may be an alternate method for SMA screening by NBS programs, providing a multiplex testing platform on which potentially hundreds of inherited conditions could be simultaneously tested.
ABSTRACT
The adipocyte fatty acid-binding protein aP2 regulates systemic glucose and lipid metabolism. We report that aP2, in addition to being abundantly expressed by adipocytes, is also expressed by human airway epithelial cells and shows a striking upregulation following stimulation of epithelial cells with the Th2 cytokines IL-4 and IL-13. Regulation of aP2 mRNA expression by Th2 cytokines was highly dependent on STAT6, a transcription factor with a major regulatory role in allergic inflammation. We examined aP2-deficient mice in a model of allergic airway inflammation and found that infiltration of leukocytes, especially eosinophils, into the airways was highly dependent on aP2 function. T cell priming was unaffected by aP2 deficiency, suggesting that aP2 was acting locally within the lung, and analysis of bone marrow chimeras implicated non-hematopoietic cells, most likely bronchial epithelial cells, as the site of action of aP2 in allergic airway inflammation. Thus, aP2 regulates allergic airway inflammation and may provide a link between fatty acid metabolism and asthma.
Subject(s)
Adipocytes/physiology , Asthma/immunology , Fatty Acid-Binding Proteins/metabolism , Hypersensitivity/immunology , Inflammation/physiopathology , 3T3 Cells , Adipocytes/immunology , Animals , Bronchi/physiology , Cell Culture Techniques , Disease Models, Animal , Fatty Acid-Binding Proteins/deficiency , Fatty Acid-Binding Proteins/genetics , Fatty Acids/metabolism , Interleukin-12/immunology , Interleukin-4/immunology , Mice , Mice, Knockout , RNA, Messenger/genetics , Respiratory Mucosa/physiology , Th2 Cells/immunology , Transcription, GeneticABSTRACT
Asthma is a chronic inflammatory disease of the airways, involving recurrent episodes of airway obstruction and wheezing. A common pathological feature in asthma is the presence of a characteristic allergic airway inflammatory response involving extensive leukocyte infiltration, mucus overproduction and airway hyper-reactivity. The pathogenesis of allergic airway inflammation is complex, involving multiple cell types such as T helper 2 cells, regulatory T cells, eosinophils, dendritic cells, mast cells, and parenchymal cells of the lung. The cellular response in allergic airway inflammation is controlled by a broad range of bioactive mediators, including IgE, cytokines and chemokines. The asthmatic allergic inflammatory response has been a particular focus of efforts to develop novel therapeutic agents. Animal models are widely used to investigate inflammatory mechanisms. Although these models are not perfect replicas of clinical asthma, such studies have led to the development of numerous novel therapeutic agents, of which some have already been successful in clinical trials.
Subject(s)
Anti-Asthmatic Agents/therapeutic use , Asthma/immunology , Disease Models, Animal , Inflammation/immunology , Mice , Animals , Anti-Asthmatic Agents/pharmacology , Anti-Inflammatory Agents/pharmacology , Anti-Inflammatory Agents/therapeutic use , Antigen Presentation , Asthma/drug therapy , Asthma/pathology , Asthma/physiopathology , Bronchial Hyperreactivity/physiopathology , Chemotaxis, Leukocyte/physiology , Cytokines/physiology , Dendritic Cells/immunology , Eosinophils/immunology , Epithelial Cells/immunology , Epithelial Cells/pathology , Humans , Immunoglobulin E/immunology , Inflammation/drug therapy , Inflammation/pathology , Inflammation/physiopathology , Mast Cells/immunology , Mice/genetics , Mice/immunology , Mice/physiology , Mice, Knockout , Mice, Transgenic , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/pathologyABSTRACT
The sensitivity and specificity of next-generation sequencing laboratory developed tests (LDTs) are typically determined by an analyte-specific approach. Analyte-specific validations use disease-specific controls to assess an LDT's ability to detect known pathogenic variants. Alternatively, a methods-based approach can be used for LDT technical validations. Methods-focused validations do not use disease-specific controls but use benchmark reference DNA that contains known variants (benign, variants of unknown significance, and pathogenic) to assess variant calling accuracy of a next-generation sequencing workflow. Recently, four whole-genome reference materials (RMs) from the National Institute of Standards and Technology (NIST) were released to standardize methods-based validations of next-generation sequencing panels across laboratories. We provide a practical method for using NIST RMs to validate multigene panels. We analyzed the utility of RMs in validating a novel newborn screening test that targets 70 genes, called NEO1. Despite the NIST RM variant truth set originating from multiple sequencing platforms, replicates, and library types, we discovered a 5.2% false-negative variant detection rate in the RM truth set genes that were assessed in our validation. We developed a strategy using complementary non-RM controls to demonstrate 99.6% sensitivity of the NEO1 test in detecting variants. Our findings have implications for laboratories or proficiency testing organizations using whole-genome NIST RMs for testing.
Subject(s)
Genome, Human , High-Throughput Nucleotide Sequencing/methods , Multigene Family , DNA Copy Number Variations , Gene Deletion , Genetic Variation , High-Throughput Nucleotide Sequencing/standards , Humans , Infant, Newborn , Infant, Newborn, Diseases/genetics , Mutagenesis, Insertional , Polymorphism, Single Nucleotide , Reference Standards , Validation Studies as TopicABSTRACT
Type 1 diabetes (T1D) is an autoimmune disease that shows familial aggregation in humans and likely has genetic determinants. Disease linkage studies have revealed many susceptibility loci for T1D in mice and humans. The mouse T1D susceptibility locus insulin-dependent diabetes susceptibility 3 (Idd3), which has a homologous genetic interval in humans, encodes cytokine genes Il2 and Il21 and regulates diabetes and other autoimmune diseases; however, the cellular and molecular mechanisms of this regulation are still being elucidated. Here we show that T cells from NOD mice produce more Il21 and less Il2 and exhibit enhanced Th17 cell generation compared with T cells from NOD.Idd3 congenic mice, which carry the protective Idd3 allele from a diabetes-resistant mouse strain. Further, APCs from NOD and NOD.Idd3 mice played a central role in this differential Th17 cell development, and IL-21 signaling in APCs was pivotal to this process. Specifically, NOD-derived APCs showed increased production of pro-Th17 mediators and dysregulation of the retinoic acid (RA) signaling pathway compared with APCs from NOD.Idd3 and NOD.Il21r-deficient mice. These data suggest that the protective effect of the Idd3 locus is due, in part, to differential RA signaling in APCs and that IL-21 likely plays a role in this process. Thus, we believe APCs provide a new candidate for therapeutic intervention in autoimmune diseases.
Subject(s)
Antigen-Presenting Cells/immunology , Diabetes Mellitus, Type 1/genetics , Diabetes Mellitus, Type 1/immunology , Interleukins/metabolism , Th17 Cells/immunology , Animals , CD11b Antigen/metabolism , Cell Differentiation/immunology , Diabetes Mellitus, Type 1/metabolism , Female , Gene Expression Profiling , Genetic Predisposition to Disease , Humans , Interleukin-2/genetics , Interleukin-2/metabolism , Interleukin-21 Receptor alpha Subunit/deficiency , Interleukin-21 Receptor alpha Subunit/genetics , Interleukin-21 Receptor alpha Subunit/metabolism , Interleukins/genetics , Mice , Mice, Congenic , Mice, Inbred NOD , Mice, Knockout , Signal Transduction/immunology , Tretinoin/metabolismABSTRACT
The fatty acid-binding protein (FABP) family consists of a number of conserved cytoplasmic proteins with roles in intracellular lipid transport, storage, and metabolism. Examination of a comprehensive leukocyte gene expression database revealed strong expression of the adipocyte FABP aP2 in human monocyte-derived dendritic cells (DCs). We isolated bone marrow-derived DC from aP2-deficient mice, and showed that expression of DC cytokines including IL-12 and TNF was significantly impaired in these cells. Degradation of IkappaBalpha was also impaired in aP2-deficient DCs, indicative of reduced signaling through the IkappaB kinase-NF-kappaB pathway. The cytokine defect was selective because there was no effect on Ag uptake or expression of MHC class II, CD40, CD80, or CD86. In an MLR, aP2-deficient DCs stimulated markedly lower T cell proliferation and cytokine production than did wild-type DCs. Moreover, aP2-deficient mice immunized with keyhole limpet hemocyanin/CFA showed reduced production of IFN-gamma by restimulated draining lymph node cells, suggesting a similar defect in DC function in vivo. Similarly, infection of aP2-deficient mice with the natural mouse pathogen ectromelia virus resulted in substantially lower production of IFN-gamma by CD8+ T cells. Thus, FABP aP2 plays an important role in DC function and T cell priming, and provides an additional link between metabolic processes and the regulation of immune responses.
Subject(s)
Dendritic Cells/immunology , Fatty Acid-Binding Proteins/immunology , Lymphocyte Activation/immunology , T-Lymphocytes/immunology , Animals , Blotting, Western , Bone Marrow Cells/immunology , Bone Marrow Cells/metabolism , Dendritic Cells/metabolism , Fatty Acid-Binding Proteins/metabolism , Gene Expression , I-kappa B Proteins/metabolism , Interleukin-12/metabolism , Lymphocyte Culture Test, Mixed , Mice , NF-KappaB Inhibitor alpha , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain Reaction , T-Lymphocytes/metabolism , Tumor Necrosis Factor-alpha/metabolismABSTRACT
B cell-activating factor belonging to the TNF family (BAFF; BLyS) is a critical regulator of B cell maturation and survival, and its overexpression in BAFF transgenic (Tg) mice results in the development of autoimmune disorders. BAFF also affects T cell function through binding to one of the BAFF receptors, BAFF-R. Using BAFF Tg mice, we examined a typical Th1-mediated response, the cutaneous delayed-type hypersensitivity reaction, and found a much greater degree of paw swelling and inflammation than in control mice. Importantly, delayed-type hypersensitivity scores correlated directly with BAFF levels in serum. Conversely, in a Th2-mediated model of allergic airway inflammation, BAFF Tg mice were largely protected and showed markedly reduced Ag-specific T cell proliferation and eosinophil infiltration associated with the airways. Thus, local and/or systemically distributed BAFF affects Th1 and Th2 responses and impacts on the course of some T cell-mediated inflammatory reactions. Our results are consistent with the idea that BAFF augments T cell as well as B cell responses, particularly Th1-type responses. Results in BAFF Tg mice may reflect the situation in certain autoimmune patients or virally infected individuals, because BAFF levels in blood are comparable.