ABSTRACT
Clinical benefits of cytokine blockade in ileal Crohn's disease (iCD) are limited to a subset of patients. Here, we applied single-cell technologies to iCD lesions to address whether cellular heterogeneity contributes to treatment resistance. We found that a subset of patients expressed a unique cellular module in inflamed tissues that consisted of IgG plasma cells, inflammatory mononuclear phagocytes, activated T cells, and stromal cells, which we named the GIMATS module. Analysis of ligand-receptor interaction pairs identified a distinct network connectivity that likely drives the GIMATS module. Strikingly, the GIMATS module was also present in a subset of patients in four independent iCD cohorts (n = 441), and its presence at diagnosis correlated with failure to achieve durable corticosteroid-free remission upon anti-TNF therapy. These results emphasize the limitations of current diagnostic assays and the potential for single-cell mapping tools to identify novel biomarkers of treatment response and tailored therapeutic opportunities.
Subject(s)
Crohn Disease/therapy , Cytokines/immunology , Intestines/pathology , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Crohn Disease/immunology , Crohn Disease/pathology , Humans , Immunotherapy/methods , Phagocytes/pathology , Single-Cell Analysis , Stromal Cells/pathology , T-Lymphocytes/pathologyABSTRACT
Crohn's disease is a chronic inflammatory intestinal disease that is frequently accompanied by aberrant healing and stricturing complications. Crosstalk between activated myeloid and stromal cells is critical in the pathogenicity of Crohn's disease1,2, and increases in intravasating monocytes are correlated with a lack of response to anti-TNF treatment3. The risk alleles with the highest effect on Crohn's disease are loss-of-function mutations in NOD24,5, which increase the risk of stricturing6. However, the mechanisms that underlie pathogenicity driven by NOD2 mutations and the pathways that might rescue a lack of response to anti-TNF treatment remain largely uncharacterized. Here we use direct ex vivo analyses of patients who carry risk alleles of NOD2 to show that loss of NOD2 leads to dysregulated homeostasis of activated fibroblasts and macrophages. CD14+ peripheral blood mononuclear cells from carriers of NOD2 risk alleles produce cells that express high levels of collagen, and elevation of conserved signatures is observed in nod2-deficient zebrafish models of intestinal injury. The enrichment of STAT3 regulation and gp130 ligands in activated fibroblasts and macrophages suggested that gp130 blockade might rescue the activated program in NOD2-deficient cells. We show that post-treatment induction of the STAT3 pathway is correlated with a lack of response to anti-TNF treatment in patients, and demonstrate in vivo in zebrafish the amelioration of the activated myeloid-stromal niche using the specific gp130 inhibitor bazedoxifene. Our results provide insights into NOD2-driven fibrosis in Crohn's disease, and suggest that gp130 blockade may benefit some patients with Crohn's disease-potentially as a complement to anti-TNF therapy.
Subject(s)
Crohn Disease/metabolism , Cytokine Receptor gp130/metabolism , Myeloid Cells/cytology , Nod2 Signaling Adaptor Protein/metabolism , Stromal Cells/cytology , Alleles , Animals , Collagen/metabolism , Cytokine Receptor gp130/antagonists & inhibitors , Disease Models, Animal , Female , Fibroblasts/cytology , Fibroblasts/metabolism , Humans , Ileitis/metabolism , Indoles/pharmacology , Interleukin-11/metabolism , Lipopolysaccharide Receptors/metabolism , Macrophages/cytology , Macrophages/metabolism , Male , Myeloid Cells/metabolism , Receptor, Platelet-Derived Growth Factor alpha/metabolism , STAT3 Transcription Factor/metabolism , Stromal Cells/metabolism , WT1 Proteins/metabolism , Zebrafish , Zebrafish Proteins/metabolismABSTRACT
Reversible infantile respiratory chain deficiency (RIRCD) is a rare mitochondrial myopathy leading to severe metabolic disturbances in infants, which recover spontaneously after 6-months of age. RIRCD is associated with the homoplasmic m.14674T>C mitochondrial DNA mutation; however, only ~ 1/100 carriers develop the disease. We studied 27 affected and 15 unaffected individuals from 19 families and found additional heterozygous mutations in nuclear genes interacting with mt-tRNAGlu including EARS2 and TRMU in the majority of affected individuals, but not in healthy carriers of m.14674T>C, supporting a digenic inheritance. Our transcriptomic and proteomic analysis of patient muscle suggests a stepwise mechanism where first, the integrated stress response associated with increased FGF21 and GDF15 expression enhances the metabolism modulated by serine biosynthesis, one carbon metabolism, TCA lipid oxidation and amino acid availability, while in the second step mTOR activation leads to increased mitochondrial biogenesis. Our data suggest that the spontaneous recovery in infants with digenic mutations may be modulated by the above described changes. Similar mechanisms may explain the variable penetrance and tissue specificity of other mtDNA mutations and highlight the potential role of amino acids in improving mitochondrial disease.
Subject(s)
Mitochondrial Diseases/genetics , Mitochondrial Diseases/metabolism , Mitochondrial Myopathies/genetics , Mitochondrial Myopathies/metabolism , Adolescent , Cell Line , DNA, Mitochondrial/genetics , Female , Gene Expression , Humans , Infant , Male , Mitochondria/metabolism , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Mutation , Pedigree , Proteomics , Quadriceps Muscle/metabolism , tRNA Methyltransferases/genetics , tRNA Methyltransferases/metabolismABSTRACT
An important goal of clinical genomics is to be able to estimate the risk of adverse disease outcomes. Between 5% and 10% of individuals with ulcerative colitis (UC) require colectomy within 5 years of diagnosis, but polygenic risk scores (PRSs) utilizing findings from genome-wide association studies (GWASs) are unable to provide meaningful prediction of this adverse status. By contrast, in Crohn disease, gene expression profiling of GWAS-significant genes does provide some stratification of risk of progression to complicated disease in the form of a transcriptional risk score (TRS). Here, we demonstrate that a measured TRS based on bulk rectal gene expression in the PROTECT inception cohort study has a positive predictive value approaching 50% for colectomy. Single-cell profiling demonstrates that the genes are active in multiple diverse cell types from both the epithelial and immune compartments. Expression quantitative trait locus (QTL) analysis identifies genes with differential effects at baseline and week 52 follow-up, but for the most part, differential expression associated with colectomy risk is independent of local genetic regulation. Nevertheless, a predicted polygenic transcriptional risk score (PPTRS) derived by summation of transcriptome-wide association study (TWAS) effects identifies UC-affected individuals at 5-fold elevated risk of colectomy with data from the UK Biobank population cohort studies, independently replicated in an NIDDK-IBDGC dataset. Prediction of gene expression from relatively small transcriptome datasets can thus be used in conjunction with TWASs for stratification of risk of disease complications.
Subject(s)
Colectomy/statistics & numerical data , Colitis, Ulcerative/surgery , Crohn Disease/surgery , Quantitative Trait Loci , Transcriptome , Biological Specimen Banks , Cohort Studies , Colitis, Ulcerative/complications , Colitis, Ulcerative/diagnosis , Colitis, Ulcerative/genetics , Colon/metabolism , Colon/pathology , Colon/surgery , Crohn Disease/complications , Crohn Disease/diagnosis , Crohn Disease/genetics , Datasets as Topic , Disease Progression , Gene Expression Profiling , Genome-Wide Association Study , Humans , Multifactorial Inheritance , Prognosis , Risk Assessment , United KingdomABSTRACT
OBJECTIVE: Loss-of-function mutations in genes generating reactive oxygen species (ROS), such as NOX1, are associated with IBD. Mechanisms whereby loss of ROS drive IBD are incompletely defined. DESIGN: ROS measurements and single-cell transcriptomics were performed on colonoids stratified by NOX1 genotype and TNFα stimulation. Clustering of epithelial cells from human UC (inflamed and uninflamed) scRNASeq was performed. Validation of M cell induction was performed by immunohistochemistry using UEA1 (ulex europaeus agglutin-1 lectin) and in vivo with DSS injury. RESULTS: TNFα induces ROS production more in NOX1-WT versus NOX1-deficient murine colonoids under a range of Wnt-mediated and Notch-mediated conditions. scRNASeq from inflamed and uninflamed human colitis versus TNFα stimulated, in vitro colonoids defines substantially shared, induced transcription factors; NOX1-deficient colonoids express substantially lower levels of STAT3 (signal transducer and activator of transcription 3), CEBPD (CCAAT enhancer-binding protein delta), DNMT1 (DNA methyltransferase) and HIF1A (hypoxia-inducible factor) baseline. Subclustering unexpectedly showed marked TNFα-mediated induction of M cells (sentinel cells overlying lymphoid aggregates) in NOX1-deficient colonoids. M cell induction by UEA1 staining is rescued with H2O2 and paraquat, defining extra- and intracellular ROS roles in maintenance of LGR5+ stem cells. DSS injury demonstrated GP2 (glycoprotein-2), basal lymphoplasmacytosis and UEA1 induction in NOX1-deficiency. Principal components analyses of M cell genes and decreased DNMT1 RNA velocity correlate with UC inflammation. CONCLUSIONS: NOX1 deficiency plus TNFα stimulation contribute to colitis through dysregulation of the stem cell niche and altered cell differentiation, enhancing basal lymphoplasmacytosis. Our findings prioritise ROS modulation for future therapies.
Subject(s)
Colitis , Inflammatory Bowel Diseases , Mice , Humans , Animals , Reactive Oxygen Species/metabolism , Tumor Necrosis Factor-alpha/adverse effects , M Cells , NADPH Oxidase 1/genetics , NADPH Oxidase 1/metabolism , Hydrogen Peroxide/adverse effects , Colitis/chemically inducedABSTRACT
BACKGROUND AND AIMS: Polygenic risk scores (PRS) may soon be used to predict inflammatory bowel disease (IBD) risk in prevention efforts. We leveraged exome-sequence and single nucleotide polymorphism (SNP) array data from 29,358 individuals in the multiethnic, randomly ascertained health system-based BioMe biobank to define effects of common and rare IBD variants on disease prediction and pathophysiology. METHODS: PRS were calculated from European, African American, and Ashkenazi Jewish (AJ) reference case-control studies, and a meta-GWAS run using all three association datasets. PRS were then combined using regression to assess which combination of scores best predicted IBD status in European, AJ, Hispanic, and African American cohorts in BioMe. Additionally, rare variants were assessed in genes associated with very early-onset IBD (VEO-IBD), by estimating genetic penetrance in each BioMe population. RESULTS: Combining risk scores based on association data from distinct ancestral populations improved IBD prediction for every population in BioMe and significantly improved prediction among European ancestry UK Biobank individuals. Lower predictive power for non-Europeans was observed, reflecting in part substantially lower African IBD case-control reference sizes. We replicated associations for two VEO-IBD genes, ADAM17 and LRBA, with high dominant model penetrance in BioMe. Autosomal recessive LRBA risk alleles are associated with severe, early-onset autoimmunity; we show that heterozygous carriage of an African-predominant LRBA protein-altering allele is associated with significantly decreased LRBA and CTLA-4 expression with T-cell activation. CONCLUSIONS: Greater genetic diversity in African populations improves prediction across populations, and generalizes some VEO-IBD genes. Increasing African American IBD case-collections should be prioritized to reduce health disparities and enhance pathophysiological insight.
Subject(s)
Black or African American/genetics , Colitis, Ulcerative/genetics , Crohn Disease/genetics , Hispanic or Latino/genetics , Jews/genetics , Multifactorial Inheritance , Penetrance , Polymorphism, Single Nucleotide , White People/genetics , Age of Onset , Case-Control Studies , Colitis, Ulcerative/diagnosis , Colitis, Ulcerative/ethnology , Crohn Disease/diagnosis , Crohn Disease/ethnology , Europe/epidemiology , Genetic Predisposition to Disease , Genome-Wide Association Study , Humans , Phenotype , Prevalence , Race Factors , Risk Assessment , Risk Factors , United States/epidemiologyABSTRACT
BACKGROUND & AIMS: Recent literature has implicated a key role for mast cells in murine models of colonic inflammation, but their role in human ulcerative colitis (UC) is not well established. A major advance has been the identification of mrgprb2 (human orthologue, MRGPX2) as mediating IgE-independent mast cell activation. We sought to define mechanisms of mast cell activation and MRGPRX2 in human UC. METHODS: Colon tissues were collected from patients with UC for bulk RNA sequencing and lamina propria cells were isolated for MRGPRX2 activation studies and single-cell RNA sequencing. Genetic association of all protein-altering G-protein coupled receptor single-nucleotide polymorphism was performed in an Ashkenazi Jewish UC case-control cohort. Variants of MRGPRX2 were transfected into Chinese hamster ovary (CHO) and human mast cell (HMC) 1.1 cells to detect genotype-dependent effects on ß-arrestin recruitment, IP-1 accumulation, and phosphorylated extracellular signal-regulated kinase. RESULTS: Mast cell-specific mediators and adrenomedullin (proteolytic precursor of PAMP-12, an MRGPRX2 agonist) are up-regulated in inflamed compared to uninflamed UC. MRGPRX2 stimulation induces carboxypeptidase secretion from inflamed UC. Of all protein-altering GPCR alleles, a unique variant of MRGPRX2, Asn62Ser, was most associated with and was bioinformatically predicted to alter arrestin recruitment. We validated that the UC protective serine allele enhances ß-arrestin recruitment, decreases IP-1, and increases phosphorylated extracellular signal-regulated kinase with MRGPRX2 agonists. Single-cell RNA sequencing defines that adrenomedullin is expressed by activated fibroblasts and epithelial cells and that interferon gamma is a key upstream regulator of mast cell gene expression. CONCLUSION: Inflamed UC regions are distinguished by MRGPRX2-mediated activation of mast cells, with decreased activation observed with a UC-protective genetic variant. These results define cell modules of UC activation and a new therapeutic target.
Subject(s)
Cell Degranulation , Colitis, Ulcerative/metabolism , Colon/metabolism , Mast Cells/metabolism , Nerve Tissue Proteins/metabolism , Receptors, G-Protein-Coupled/metabolism , Receptors, Neuropeptide/metabolism , Adrenomedullin/genetics , Adrenomedullin/metabolism , Animals , CHO Cells , Case-Control Studies , Colitis, Ulcerative/genetics , Colitis, Ulcerative/immunology , Colon/immunology , Cricetulus , Extracellular Signal-Regulated MAP Kinases/metabolism , Genetic Variation , Humans , Inositol Phosphates/metabolism , Ligands , Mast Cells/immunology , Nerve Tissue Proteins/genetics , Phosphorylation , Receptors, G-Protein-Coupled/genetics , Receptors, Neuropeptide/genetics , beta-Arrestin 2/genetics , beta-Arrestin 2/metabolismABSTRACT
Genome-wide association studies have identified ~170 loci associated with Crohn's disease (CD) and defining which genes drive these association signals is a major challenge. The primary aim of this study was to define which CD locus genes are most likely to be disease related. We developed a gene prioritization regression model (GPRM) by integrating complementary mRNA expression datasets, including bulk RNA-Seq from the terminal ileum of 302 newly diagnosed, untreated CD patients and controls, and in stimulated monocytes. Transcriptome-wide association and co-expression network analyses were performed on the ileal RNA-Seq datasets, identifying 40 genome-wide significant genes. Co-expression network analysis identified a single gene module, which was substantially enriched for CD locus genes and most highly expressed in monocytes. By including expression-based and epigenetic information, we refined likely CD genes to 2.5 prioritized genes per locus from an average of 7.8 total genes. We validated our model structure using cross-validation and our prioritization results by protein-association network analyses, which demonstrated significantly higher CD gene interactions for prioritized compared with non-prioritized genes. Although individual datasets cannot convey all of the information relevant to a disease, combining data from multiple relevant expression-based datasets improves prediction of disease genes and helps to further understanding of disease pathogenesis.
Subject(s)
Crohn Disease/genetics , Monocytes/pathology , Sequence Analysis, DNA/methods , Adolescent , Algorithms , Case-Control Studies , Child , Child, Preschool , Crohn Disease/metabolism , Female , Gene Regulatory Networks/genetics , Genetic Predisposition to Disease/genetics , Genome-Wide Association Study , Humans , Male , Monocytes/metabolism , Polymorphism, Single Nucleotide/genetics , Quantitative Trait Loci/genetics , Software , Transcriptome/geneticsABSTRACT
The expressivity of Mendelian diseases can be influenced by factors independent from the pathogenic mutation: in Duchenne muscular dystrophy (DMD), for instance, age at loss of ambulation (LoA) varies between individuals whose DMD mutations all abolish dystrophin expression. This suggests the existence of trans-acting variants in modifier genes. Common single nucleotide polymorphisms (SNPs) in candidate genes (SPP1, encoding osteopontin, and LTBP4, encoding latent transforming growth factor ß [TGFß]-binding protein 4) have been established as DMD modifiers. We performed a genome-wide association study of age at LoA in a sub-cohort of European or European American ancestry (n = 109) from the Cooperative International Research Group Duchenne Natural History Study (CINRG-DNHS). We focused on protein-altering variants (Exome Chip) and included glucocorticoid treatment as a covariate. As expected, due to the small population size, no SNPs displayed an exome-wide significant p value (< 1.8 × 10-6). Subsequently, we prioritized 438 SNPs in the vicinities of 384 genes implicated in DMD-related pathways, i.e., the nuclear-factor-κB and TGFß pathways. The minor allele at rs1883832, in the 5'-untranslated region of CD40, was associated with earlier LoA (p = 3.5 × 10-5). This allele diminishes the expression of CD40, a co-stimulatory molecule for T cell polarization. We validated this association in multiple independent DMD cohorts (United Dystrophinopathy Project, Bio-NMD, and Padova, total n = 660), establishing this locus as a DMD modifier. This finding points to cell-mediated immunity as a relevant pathogenetic mechanism and potential therapeutic target in DMD.
Subject(s)
CD40 Antigens/genetics , Muscular Dystrophy, Duchenne/genetics , NF-kappa B/genetics , Polymorphism, Single Nucleotide , Transforming Growth Factor beta/genetics , Adolescent , Alleles , CD40 Antigens/metabolism , Case-Control Studies , Child , Dystrophin/genetics , Dystrophin/metabolism , Exons , Genes, Modifier , Genome-Wide Association Study , Glucocorticoids/pharmacology , Humans , Latent TGF-beta Binding Proteins/genetics , Latent TGF-beta Binding Proteins/metabolism , Mutation , NF-kappa B/metabolism , Osteopontin/genetics , Osteopontin/metabolism , Transforming Growth Factor beta/metabolism , White People/geneticsABSTRACT
In this paper, we present miRTarVis+, a Web-based interactive visual analytics tool for miRNA target predictions and integrative analyses of multiple prediction results. Various microRNA (miRNA) target prediction algorithms have been developed to improve sequence-based miRNA target prediction by exploiting miRNA-mRNA expression profile data. There are also a few analytics tools to help researchers predict targets of miRNAs. However, there still is a need for improving the performance for miRNA prediction algorithms and more importantly for interactive visualization tools for an integrative analysis of multiple prediction results. miRTarVis+ has an intuitive interface to support the analysis pipeline of load, filter, predict, and visualize. It can predict targets of miRNA by adopting Bayesian inference and maximal information-based nonparametric exploration (MINE) analyses as well as conventional correlation and mutual information analyses. miRTarVis+ supports an integrative analysis of multiple prediction results by providing an overview of multiple prediction results and then allowing users to examine a selected miRNA-mRNA network in an interactive treemap and node-link diagram. To evaluate the effectiveness of miRTarVis+, we conducted two case studies using miRNA-mRNA expression profile data of asthma and breast cancer patients and demonstrated that miRTarVis+ helps users more comprehensively analyze targets of miRNA from miRNA-mRNA expression profile data. miRTarVis+ is available at http://hcil.snu.ac.kr/research/mirtarvisplus.
Subject(s)
Asthma/genetics , Breast Neoplasms/genetics , MicroRNAs/genetics , RNA, Messenger/genetics , Sequence Analysis, RNA/methods , User-Computer Interface , Algorithms , Asthma/diagnosis , Asthma/metabolism , Asthma/pathology , Base Sequence , Bayes Theorem , Binding Sites , Breast Neoplasms/diagnosis , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Female , Gene Expression Profiling , Gene Expression Regulation , Humans , Internet , MicroRNAs/metabolism , RNA, Messenger/metabolismABSTRACT
Epigenomic regulation of the transcriptome by DNA methylation and posttranscriptional gene silencing by miRNAs are potential environmental modulators of skeletal muscle plasticity to chronic exercise in healthy and diseased populations. We utilized transcriptome networks to connect exercise-induced differential methylation and miRNA with functional skeletal muscle plasticity. Biopsies of the vastus lateralis were collected from middle-aged Polynesian men and women with morbid obesity (44 kg/m(2) ± 10) and Type 2 diabetes before and following 16 wk of resistance (n = 9) or endurance training (n = 8). Longitudinal transcriptome, methylome, and microRNA (miRNA) responses were obtained via microarray, filtered by novel effect-size based false discovery rate probe selection preceding bioinformatic interrogation. Metabolic and microvascular transcriptome topology dominated the network landscape following endurance exercise. Lipid and glucose metabolism modules were connected to: microRNA (miR)-29a; promoter region hypomethylation of nuclear receptor factor (NRF1) and fatty acid transporter (SLC27A4), and hypermethylation of fatty acid synthase, and to exon hypomethylation of 6-phosphofructo-2-kinase and Ser/Thr protein kinase. Directional change in the endurance networks was validated by lower intramyocellular lipid, increased capillarity, GLUT4, hexokinase, and mitochondrial enzyme activity and proteome. Resistance training also lowered lipid and increased enzyme activity and caused GLUT4 promoter hypomethylation; however, training was inconsequential to GLUT4, capillarity, and metabolic transcriptome. miR-195 connected to negative regulation of vascular development. To conclude, integrated molecular network modelling revealed differential DNA methylation and miRNA expression changes occur in skeletal muscle in response to chronic exercise training that are most pronounced with endurance training and topographically associated with functional metabolic and microvascular plasticity relevant to diabetes rehabilitation.
Subject(s)
DNA Methylation/genetics , Diabetes Mellitus, Type 2/genetics , Exercise , Gene Regulatory Networks , MicroRNAs/genetics , Muscle, Skeletal/pathology , Obesity/genetics , Diabetes Mellitus, Type 2/complications , Epigenesis, Genetic , Female , Gene Expression Regulation , Glucose/metabolism , Humans , Lipid Metabolism , Male , MicroRNAs/metabolism , Middle Aged , Muscle, Skeletal/blood supply , Muscle, Skeletal/ultrastructure , Obesity/complications , Phenotype , Physical Endurance/genetics , Proteomics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Resistance Training , Transcriptome/geneticsABSTRACT
Inflammatory bowel disease (IBD) is a group of chronic digestive tract inflammatory conditions whose genetic etiology is still poorly understood. The incidence of IBD is particularly high among Ashkenazi Jews. Here, we identify 8 novel and plausible IBD-causing genes from the exomes of 4453 genetically identified Ashkenazi Jewish IBD cases (1734) and controls (2719). Various biological pathway analyses are performed, along with bulk and single-cell RNA sequencing, to demonstrate the likely physiological relatedness of the novel genes to IBD. Importantly, we demonstrate that the rare and high impact genetic architecture of Ashkenazi Jewish adult IBD displays significant overlap with very early onset-IBD genetics. Moreover, by performing biobank phenome-wide analyses, we find that IBD genes have pleiotropic effects that involve other immune responses. Finally, we show that polygenic risk score analyses based on genome-wide high impact variants have high power to predict IBD susceptibility.
Subject(s)
Inflammatory Bowel Diseases , Jews , Adult , Humans , Jews/genetics , Exome/genetics , Inflammatory Bowel Diseases/genetics , Risk Assessment , Genetic Predisposition to DiseaseABSTRACT
The mechanisms underlying liver fibrosis are multifaceted and remain elusive with no approved antifibrotic treatments available. The adult zebrafish has been an underutilized tool to study liver fibrosis. We aimed to characterize the single-cell transcriptome of the adult zebrafish liver to determine its utility as a model for studying liver fibrosis. We used single-cell RNA sequencing (scRNA-seq) of adult zebrafish liver to study the molecular and cellular dynamics at a single-cell level. We performed a comparative analysis to scRNA-seq of human liver with a focus on hepatic stellate cells (HSCs), the driver cells in liver fibrosis. scRNA-seq reveals transcriptionally unique populations of hepatic cell types that comprise the zebrafish liver. Joint clustering with human liver scRNA-seq data demonstrates high conservation of transcriptional profiles and human marker genes in zebrafish. Human and zebrafish HSCs show conservation of transcriptional profiles, and we uncover collectin subfamily member 11 (colec11) as a novel, conserved marker for zebrafish HSCs. To demonstrate the power of scRNA-seq to study liver fibrosis using zebrafish, we performed scRNA-seq on our zebrafish model of a pediatric liver disease with mutation in mannose phosphate isomerase (MPI) and characteristic early liver fibrosis. We found fibrosis signaling pathways and upstream regulators conserved across MPI-depleted zebrafish and human HSCs. CellPhoneDB analysis of zebrafish transcriptome identified neuropilin 1 as a potential driver of liver fibrosis. Conclusion: This study establishes the first scRNA-seq atlas of the adult zebrafish liver, highlights the high degree of similarity to human liver, and strengthens its value as a model to study liver fibrosis.
Subject(s)
Transcriptome , Zebrafish , Animals , Child , Humans , Liver Cirrhosis/genetics , Mannose-6-Phosphate Isomerase , Phenotype , Transcriptome/genetics , Zebrafish/geneticsABSTRACT
BACKGROUND AND AIMS: Crohn's disease [CD] recurrence following ileocolic resection [ICR] is common. We sought to identify blood-based biomarkers associated with CD recurrence. METHODS: CD patients undergoing ICR were recruited across six centres. Serum samples were obtained at post-operative colonoscopy. A multiplex immunoassay was used to analyse 92 inflammation-related proteins [Olink Proteomics]. Bayesian analysis was used to identify proteins associated with increasing Rutgeerts score. Identified proteins were used in receiver operating characteristic [ROC] analysis to examine the ability to identify CD recurrence [Rutgeerts score ≥i2]. Existing single cell data were interrogated to further elucidate the role of the identified proteins. RESULTS: Data from 276 colonoscopies in 213 patients were available. Median time from surgery to first and second colonoscopy was 7 (interquartile range [IQR] 6-9) and 19 [IQR 16-23] months, respectively. Disease recurrence was evident at 60 [30%] first and 36 [49%] second colonoscopies. Of 14 proteins significantly associated with Rutgeerts score, the strongest signal was seen for CXCL9 and MMP1. Among patients on anti-tumour necrosis factor drugs, CXCL9 and CXCL11 were most strongly associated with Rutgeerts score. Both are CXCR3 ligands. Incorporation of identified proteins into ROC analysis improved the ability to identify disease recurrence as compared to C-reactive protein alone: area under the curve [AUC] 0.75 (95% confidence interval [CI]: 0.66-0.82] vs 0.64 [95% CI 0.56-0.72], p = 0.012. Single cell transcriptomic data provide evidence that innate immune cells are the primary source of the identified proteins. CONCLUSIONS: CXCR3 ligands are associated with CD recurrence following ICR. Incorporation of novel blood-based candidate biomarkers may aid in identification of CD recurrence.
Subject(s)
Crohn Disease , Bayes Theorem , Biomarkers/metabolism , Colonoscopy , Crohn Disease/diagnosis , Crohn Disease/metabolism , Crohn Disease/surgery , Humans , Ileum/pathology , Receptors, CXCR3 , Recurrence , Retrospective StudiesABSTRACT
Genome-wide association studies (GWASs) have identified hundreds of loci associated with Crohn's disease (CD). However, as with all complex diseases, robust identification of the genes dysregulated by noncoding variants typically driving GWAS discoveries has been challenging. Here, to complement GWASs and better define actionable biological targets, we analyzed sequence data from more than 30,000 patients with CD and 80,000 population controls. We directly implicate ten genes in general onset CD for the first time to our knowledge via association to coding variation, four of which lie within established CD GWAS loci. In nine instances, a single coding variant is significantly associated, and in the tenth, ATG4C, we see additionally a significantly increased burden of very rare coding variants in CD cases. In addition to reiterating the central role of innate and adaptive immune cells as well as autophagy in CD pathogenesis, these newly associated genes highlight the emerging role of mesenchymal cells in the development and maintenance of intestinal inflammation.
Subject(s)
Crohn Disease , Crohn Disease/genetics , Genetic Predisposition to Disease , Genome-Wide Association Study , Humans , Polymorphism, Single Nucleotide/geneticsABSTRACT
Duchenne muscular dystrophy is initiated by dystrophin deficiency, but downstream pathophysiological pathways such as membrane instability, NFĸB activation, mitochondrial dysfunction, and induction of TGFß fibrosis pathways are thought to drive the disability. Dystrophin replacement strategies are hopeful for addressing upstream dystrophin deficiency; however, all methods to date use semi-functional dystrophin proteins that are likely to trigger downstream pathways. Thus, combination therapies that can target multiple downstream pathways are important in treating DMD, even for dystrophin-replacement strategies. We sought to define blood pharmacodynamic biomarkers of drug response in the mdx mouse model of Duchenne muscular dystrophy using a series of repurposed drugs. Four-week-old mdx mice were treated for four weeks with four different drugs singly and in combination: vehicle, prednisolone, vamorolone, rituximab, ß-aminoisobutyric acid (BAIBA) (11 treatment groups; n = 6/group). Blood was collected via cardiac puncture at study termination, and proteomic profiling was carried out using SOMAscan aptamer panels (1,310 proteins assayed). Prednisolone was tested alone and in combination with other drugs. It was found to have a good concordance of prednisolone-responsive biomarkers (56 increased by prednisolone, 39 decreased) focused on NFκB and TGFß cascades. Vamorolone shared 45 (80%) of increased biomarkers and 13 (33%) of decreased biomarkers with prednisolone. Comparison of published human corticosteroid-responsive biomarkers to our mdx data showed 14% (3/22) concordance between mouse and human. Rituximab showed fewer drug-associated biomarkers, with the most significant being human IgG. On the other hand, BAIBA treatment (high and low dose) showed a drug-associated increase in 40 serum proteins and decreased 5 serum proteins. Our results suggest that a biomarker approach could be employed for assessing drug combinations in both mouse and human studies.
Subject(s)
Muscular Dystrophy, Animal/drug therapy , Muscular Dystrophy, Duchenne/drug therapy , Aminoisobutyric Acids/therapeutic use , Animals , Biomarkers , Disease Models, Animal , Drug Repositioning , Male , Mice , Mice, Inbred mdx , Prednisolone/therapeutic use , Pregnadienediols/therapeutic use , Rituximab/therapeutic useABSTRACT
Genome-wide association studies have identified over 200 genomic loci associated with inflammatory bowel disease (IBD). High-effect risk alleles define key roles for genes involved in bacterial response and innate defense. More high-throughput in vivo systems are required to rapidly evaluate therapeutic agents. We visualize, in zebrafish, the effects on epithelial barrier function and intestinal autophagy of one-course and repetitive injury. Repetitive injury induces increased mortality, impaired recovery of intestinal barrier function, failure to contain bacteria within the intestine and impaired autophagy. Prostaglandin E2 (PGE2) administration protected against injury by enhancing epithelial barrier function and limiting systemic infection. Effects of IBD therapeutic agents were defined: mesalamine showed protective features during injury, whereas 6-mercaptopurine displayed marked induction of autophagy during recovery. Given the highly conserved nature of innate defense in zebrafish, it represents an ideal model system with which to test established and new IBD therapies targeted to the epithelial barrier.This article has an associated First Person interview with the first author of the paper.
Subject(s)
Epithelium/pathology , Inflammatory Bowel Diseases/microbiology , Inflammatory Bowel Diseases/pathology , Intestines/injuries , Zebrafish/physiology , Acids/metabolism , Animals , Autophagy , Bacterial Proteins/metabolism , Dextran Sulfate , Dinoprostone/metabolism , Disease Models, Animal , Enterocytes/metabolism , Humans , Inflammatory Bowel Diseases/drug therapy , Intestines/pathology , Lysosomes/metabolism , Models, Biological , Mucins/metabolism , Mucus/metabolismABSTRACT
PURPOSE: Whole body insulin sensitivity (Si) typically improves after aerobic exercise training; however, individual responses can be highly variable. The purpose of this study was to use global gene expression to identify skeletal muscle genes that correlate with exercise-induced Si changes. METHODS: Longitudinal cohorts from the Studies of Targeted Risk Reduction Intervention through Defined Exercise were used as Discovery (Affymetrix) and Confirmation (Illumina) of vastus lateralis gene expression profiles. Discovery (n = 39; 21 men) and Confirmation (n = 42; 19 men) cohorts were matched for age (52 ± 8 vs 51 ± 10 yr), body mass index (30.4 ± 2.8 vs 29.7 ± 2.8 kg·m), and VËO2max (30.4 ± 2.8 vs 29.7 ± 2.8 mL·kg·min). Si was determined via intravenous glucose tolerance test pretraining and posttraining. Pearson product-moment correlation coefficients determined relationships between a) baseline and b) training-induced changes in gene expression and %ΔSi after training. RESULTS: Expression of 2454 (Discovery) and 1778 genes (Confirmation) at baseline were significantly (P < 0.05) correlated to %ΔSi; 112 genes overlapped. Pathway analyses identified Ca signaling-related transcripts in this 112-gene list. Expression changes of 1384 (Discovery) and 1288 genes (Confirmation) after training were significantly (P < 0.05) correlated to %ΔSi; 33 genes overlapped, representing contractile apparatus of skeletal and smooth muscle genes. Pyruvate dehydrogenase phosphatase regulatory subunit expression at baseline (P = 0.01, r = 0.41) and posttraining (P = 0.01, r = 0.43) were both correlated with %ΔSi. CONCLUSIONS: Exercise-induced adaptations in skeletal muscle Si are related to baseline levels of Ca-regulating transcripts, which may prime the muscle for adaptation. Relationships between %ΔSi and pyruvate dehydrogenase phosphatase regulatory, a regulatory subunit of the pyruvate dehydrogenase complex, indicate that the Si response is strongly related to key steps in metabolic regulation.
Subject(s)
Exercise/physiology , Gene Expression , Insulin Resistance/physiology , Muscle, Skeletal/enzymology , Pyruvate Dehydrogenase (Lipoamide)-Phosphatase/genetics , Calcium Signaling , Cardiovascular Diseases/prevention & control , Female , Humans , Longitudinal Studies , Male , Middle Aged , Muscle, Skeletal/metabolism , Obesity/metabolism , Risk Factors , Sedentary BehaviorABSTRACT
BACKGROUND: Innate immune responses are fine-tuned by small noncoding RNA molecules termed microRNAs (miRs) that modify gene expression in response to the environment. During acute infections, miRs can be secreted in extracellular vesicles (EV) to facilitate cell-to-cell genetic communication. The purpose of this study was to characterize the baseline population of miRs secreted in EVs in the airways of young children (airway secretory microRNAome) and examine the changes during rhinovirus (RV) infection, the most common cause of asthma exacerbations and the most important early risk factor for the development of asthma beyond childhood. METHODS: Nasal airway secretions were obtained from children (≤3 yrs. old) during PCR-confirmed RV infections (n = 10) and age-matched controls (n = 10). Nasal EVs were isolated with polymer-based precipitation and global miR profiles generated using NanoString microarrays. We validated our in vivo airway secretory miR data in an in vitro airway epithelium model using apical secretions from primary human bronchial epithelial cells (HBEC) differentiated at air-liquid interface (ALI). Bioinformatics tools were used to determine the unified (nasal and bronchial) signature airway secretory miRNAome and changes during RV infection in children. RESULTS: Multiscale analysis identified four signature miRs comprising the baseline airway secretory miRNAome: hsa-miR-630, hsa-miR-302d-3p, hsa- miR-320e, hsa-miR-612. We identified hsa-miR-155 as the main change in the baseline miRNAome during RV infection in young children. We investigated the potential biological relevance of the airway secretion of hsa-mir-155 using in silico models derived from gene datasets of experimental in vivo human RV infection. These analyses confirmed that hsa-miR-155 targetome is an overrepresented pathway in the upper airways of individuals infected with RV. CONCLUSIONS: Comparative analysis of the airway secretory microRNAome in children indicates that RV infection is associated with airway secretion of EVs containing miR-155, which is predicted in silico to regulate antiviral immunity. Further characterization of the airway secretory microRNAome during health and disease may lead to completely new strategies to treat and monitor respiratory conditions in all ages.
Subject(s)
Gene Expression Regulation , MicroRNAs/genetics , Picornaviridae Infections/genetics , Respiratory Tract Infections/genetics , Rhinovirus/physiology , Child, Preschool , Genomics , Humans , Infant , Up-RegulationABSTRACT
BACKGROUND: Molecular diagnostics in the genetic myopathies often requires testing of the largest and most complex transcript units in the human genome (DMD, TTN, NEB). Iteratively targeting single genes for sequencing has traditionally entailed high costs and long turnaround times. Exome sequencing has begun to supplant single targeted genes, but there are concerns regarding coverage and needed depth of the very large and complex genes that frequently cause myopathies. OBJECTIVE: To evaluate efficiency of next-generation sequencing technologies to provide molecular diagnostics for patients with previously undiagnosed myopathies. METHODS: We tested a targeted re-sequencing approach, using a 45 gene emulsion PCR myopathy panel, with subsequent sequencing on the Illumina platform in 94 undiagnosed patients. We compared the targeted re-sequencing approach to exome sequencing for 10 of these patients studied. RESULTS: We detected likely pathogenic mutations in 33 out of 94 patients with a molecular diagnostic rate of approximately 35%. The remaining patients showed variants of unknown significance (35/94 patients) or no mutations detected in the 45 genes tested (26/94 patients). Mutation detection rates for targeted re-sequencing vs. whole exome were similar in both methods; however exome sequencing showed better distribution of reads and fewer exon dropouts. CONCLUSIONS: Given that costs of highly parallel re-sequencing and whole exome sequencing are similar, and that exome sequencing now takes considerably less laboratory processing time than targeted re-sequencing, we recommend exome sequencing as the standard approach for molecular diagnostics of myopathies.