ABSTRACT
Analyses of transcriptional bursting from single-cell RNA-sequencing data have revealed patterns of variation and regulation in the kinetic parameters that could be inferred. Here we profiled newly transcribed (4-thiouridine-labelled) RNA across 10,000 individual primary mouse fibroblasts to more broadly infer bursting kinetics and coordination. We demonstrate that inference from new RNA profiles could separate the kinetic parameters that together specify the burst size, and that the synthesis rate (and not the transcriptional off rate) controls the burst size. Importantly, transcriptome-wide inference of transcriptional on and off rates provided conclusive evidence that RNA polymerase II transcribes genes in bursts. Recent reports identified examples of transcriptional co-bursting, yet no global analyses have been performed. The deep new RNA profiles we generated with allelic resolution demonstrated that co-bursting rarely appears more frequently than expected by chance, except for certain gene pairs, notably paralogues located in close genomic proximity. Altogether, new RNA single-cell profiling critically improves the inference of transcriptional bursting and provides strong evidence for independent transcriptional bursting of mammalian genes.
ABSTRACT
Neonatal hypoxia-ischemia (HI) is a major cause of perinatal death and long-term disabilities worldwide. Post-ischemic neuroinflammation plays a pivotal role in HI pathophysiology. In the present study, we investigated the temporal dynamics of microglia (CX3CR1GFP/+) and infiltrating macrophages (CCR2RFP/+) in the hippocampi of mice subjected to HI at postnatal day 9. Using inflammatory pathway and transcription factor (TF) analyses, we identified a distinct post-ischemic response in CCR2RFP/+ cells characterized by differential gene expression in sensome, homeostatic, matrisome, lipid metabolic, and inflammatory molecular signatures. Three days after injury, transcriptomic signatures of CX3CR1GFP/+ and CCR2RFP/+ cells isolated from hippocampi showed a partial convergence. Interestingly, microglia-specific genes in CX3CR1GFP/+ cells showed a sexual dimorphism, where expression returned to control levels in males but not in females during the experimental time frame. These results highlight the importance of further investigations on metabolic rewiring to pave the way for future interventions in asphyxiated neonates.
ABSTRACT
Idiopathic inflammatory myopathies (IIM) are rare autoimmune systemic diseases characterized by muscle weakness and the presence of muscle-infiltrating T cells. IIM represent a clinical challenge due to heterogeneity of symptoms and variability of response to immunosuppressive treatment. Here, we performed in-depth single-cell sequencing on muscle-infiltrating T cells and peripheral blood memory T cells in six patients with recently diagnosed IIM. We identified tissue resident memory T-cell (TRM ) signatures including the expression of HOBIT, XCL1 and CXCR6 in the muscle biopsies of all patients with IIM. Clonally expanded T-cell clones were mainly found among cytotoxic and TRM implying their role in the disease pathogenesis. Finally, identical expanded T-cell clones persisting at follow-up in the muscle tissue of two patients suggest their involvement in disease chronicity. Our study reveals a muscle tissue resident memory T-cell signature in patients with IIM and a transcriptomic map to identify novel therapeutic targets in IIM.
Subject(s)
Autoimmune Diseases , Myositis , Humans , T-Lymphocytes , Myositis/diagnosis , Myositis/therapy , MusclesABSTRACT
OBJECTIVE: Neonatal lupus erythematosus (NLE) may develop after transplacental transfer of maternal autoantibodies with cardiac manifestations (congenital heart block, CHB) including atrioventricular block, atrial and ventricular arrhythmias, and cardiomyopathies. The association with anti-Ro/SSA antibodies is well established, but a recurrence rate of only 12%-16% despite persisting maternal autoantibodies suggests that additional factors are required for CHB development. Here, we identify fetal genetic variants conferring risk of CHB and elucidate their effects on cardiac function. METHODS: A genome-wide association study was performed in families with at least one case of CHB. Gene expression was analysed by microarrays, RNA sequencing and PCR and protein expression by western blot, immunohistochemistry, immunofluorescence and flow cytometry. Calcium regulation and connectivity were analysed in primary cardiomyocytes and cells induced from pleuripotent stem cells. Fetal heart performance was analysed by Doppler/echocardiography. RESULTS: We identified DNAJC6 as a novel fetal susceptibility gene, with decreased cardiac expression of DNAJC6 associated with the disease risk genotype. We further demonstrate that fetal cardiomyocytes deficient in auxilin, the protein encoded by DNAJC6, have abnormal connectivity and Ca2+ homoeostasis in culture, as well as decreased cell surface expression of the Cav1.3 calcium channel. Doppler echocardiography of auxilin-deficient fetal mice revealed cardiac NLE abnormalities in utero, including abnormal heart rhythm with atrial and ventricular ectopias, as well as a prolonged atrioventricular time intervals. CONCLUSIONS: Our study identifies auxilin as the first genetic susceptibility factor in NLE modulating cardiac function, opening new avenues for the development of screening and therapeutic strategies in CHB.
Subject(s)
Atrioventricular Block , Auxilins , Animals , Antibodies, Antinuclear , Atrioventricular Block/genetics , Autoantibodies , Fetal Heart , Genome-Wide Association Study , Heart Block/congenital , Lupus Erythematosus, Systemic/congenital , MiceABSTRACT
Understanding the genetic background of complex diseases requires the expansion of studies beyond univariate associations. Therefore, it is important to use interaction assessments of risk factors in order to discover whether, and how genetic risk variants act together on disease development. The principle of interaction analysis is to explore the magnitude of the combined effect of risk factors on disease causation. In this study, we use simulations to investigate different scenarios of causation to show how the magnitude of the effect of two risk factors interact. We mainly focus on the two most commonly used interaction models, the additive and multiplicative risk scales, since there is often confusion regarding their use and interpretation. Our results show that the combined effect is multiplicative when two risk factors are involved in the same chain of events, an interaction called synergism. Synergism is often described as a deviation from additivity, which is a broader term. Our results also confirm that it is often relevant to estimate additive effect relationships, because they correspond to independent risk factors at low disease prevalence. Importantly, we evaluate the threshold of more than two required risk factors for disease causation, called the multifactorial threshold model. We found a simple mathematical relationship (square root) between the threshold and an additive-to-multiplicative linear effect scale (AMLES), where 0 corresponds to an additive effect and 1 to a multiplicative. We propose AMLES as a metric that could be used to test different effects relationships at the same time, given that it can simultaneously reveal additive, multiplicative and intermediate risk effects relationships. Finally, the utility of our simulation study was demonstrated using real data by analyzing and interpreting gene-gene interaction odds ratios from a rheumatoid arthritis case-control cohort.
Subject(s)
Arthritis, Rheumatoid/epidemiology , Arthritis, Rheumatoid/genetics , Genetic Predisposition to Disease/genetics , Models, Statistical , Polymorphism, Single Nucleotide , Alleles , Anti-Citrullinated Protein Antibodies/metabolism , Arthritis, Rheumatoid/immunology , Databases, Genetic , Europe/epidemiology , Gene Frequency , Genetic Loci , Genome-Wide Association Study , HLA-DRB1 Chains/genetics , Humans , Protein Tyrosine Phosphatase, Non-Receptor Type 22/genetics , Risk FactorsABSTRACT
Large-scale sequencing of RNA from individual cells can reveal patterns of gene, isoform and allelic expression across cell types and states1. However, current short-read single-cell RNA-sequencing methods have limited ability to count RNAs at allele and isoform resolution, and long-read sequencing techniques lack the depth required for large-scale applications across cells2,3. Here we introduce Smart-seq3, which combines full-length transcriptome coverage with a 5' unique molecular identifier RNA counting strategy that enables in silico reconstruction of thousands of RNA molecules per cell. Of the counted and reconstructed molecules, 60% could be directly assigned to allelic origin and 30-50% to specific isoforms, and we identified substantial differences in isoform usage in different mouse strains and human cell types. Smart-seq3 greatly increased sensitivity compared to Smart-seq2, typically detecting thousands more transcripts per cell. We expect that Smart-seq3 will enable large-scale characterization of cell types and states across tissues and organisms.
Subject(s)
Gene Expression Profiling/methods , RNA/analysis , Sequence Analysis, RNA/methods , Single-Cell Analysis/methods , Alleles , Animals , Humans , Mice , RNA/genetics , RNA Isoforms/analysis , RNA Isoforms/genetics , Sensitivity and Specificity , Transcriptome/geneticsABSTRACT
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
ABSTRACT
Objective- The Wnt/ß-catenin pathway orchestrates development of the blood-brain barrier, but the downstream mechanisms involved at different developmental windows and in different central nervous system (CNS) tissues have remained elusive. Approach and Results- Here, we create a new mouse model allowing spatiotemporal investigations of Wnt/ß-catenin signaling by induced overexpression of Axin1, an inhibitor of ß-catenin signaling, specifically in endothelial cells ( Axin1 iEC- OE). AOE (Axin1 overexpression) in Axin1 iEC- OE mice at stages following the initial vascular invasion of the CNS did not impair angiogenesis but led to premature vascular regression followed by progressive dilation and inhibition of vascular maturation resulting in forebrain-specific hemorrhage 4 days post-AOE. Analysis of the temporal Wnt/ß-catenin driven CNS vascular development in zebrafish also suggested that Axin1 iEC- OE led to CNS vascular regression and impaired maturation but not inhibition of ongoing angiogenesis within the CNS. Transcriptomic profiling of isolated, ß-catenin signaling-deficient endothelial cells during early blood-brain barrier-development (E11.5) revealed ECM (extracellular matrix) proteins as one of the most severely deregulated clusters. Among the 20 genes constituting the forebrain endothelial cell-specific response signature, 8 ( Adamtsl2, Apod, Ctsw, Htra3, Pglyrp1, Spock2, Ttyh2, and Wfdc1) encoded bona fide ECM proteins. This specific ß-catenin-responsive ECM signature was also repressed in Axin1 iEC- OE and endothelial cell-specific ß-catenin-knockout mice ( Ctnnb1-KOiEC) during initial blood-brain barrier maturation (E14.5), consistent with an important role of Wnt/ß-catenin signaling in orchestrating the development of the forebrain vascular ECM. Conclusions- These results suggest a novel mechanism of establishing a CNS endothelium-specific ECM signature downstream of Wnt-ß-catenin that impact spatiotemporally on blood-brain barrier differentiation during forebrain vessel development. Visual Overview- An online visual overview is available for this article.
Subject(s)
Extracellular Matrix/physiology , Prosencephalon/blood supply , Wnt Signaling Pathway/physiology , beta Catenin/physiology , Animals , Axin Protein/physiology , Blood-Brain Barrier , Male , Mice , Mice, Inbred C57BL , Signal Transduction/physiology , Vascular Remodeling , ZebrafishABSTRACT
Objective- Pathological neovascularization is crucial for progression and morbidity of serious diseases such as cancer, diabetic retinopathy, and age-related macular degeneration. While mechanisms of ongoing pathological neovascularization have been extensively studied, the initiating pathological vascular remodeling (PVR) events, which precede neovascularization remains poorly understood. Here, we identify novel molecular and cellular mechanisms of preneovascular PVR, by using the adult choriocapillaris as a model. Approach and Results- Using hypoxia or forced overexpression of VEGF (vascular endothelial growth factor) in the subretinal space to induce PVR in zebrafish and rats respectively, and by analyzing choriocapillaris membranes adjacent to choroidal neovascular lesions from age-related macular degeneration patients, we show that the choriocapillaris undergo robust induction of vascular intussusception and permeability at preneovascular stages of PVR. This PVR response included endothelial cell proliferation, formation of endothelial luminal processes, extensive vesiculation and thickening of the endothelium, degradation of collagen fibers, and splitting of existing extravascular columns. RNA-sequencing established a role for endothelial tight junction disruption, cytoskeletal remodeling, vesicle- and cilium biogenesis in this process. Mechanistically, using genetic gain- and loss-of-function zebrafish models and analysis of primary human choriocapillaris endothelial cells, we determined that HIF (hypoxia-induced factor)-1α-VEGF-A-VEGFR2 signaling was important for hypoxia-induced PVR. Conclusions- Our findings reveal that PVR involving intussusception and splitting of extravascular columns, endothelial proliferation, vesiculation, fenestration, and thickening is induced before neovascularization, suggesting that identifying and targeting these processes may prevent development of advanced neovascular disease in the future. Visual Overview- An online visual overview is available for this article.
Subject(s)
Neovascularization, Pathologic/etiology , Vascular Remodeling/physiology , Adult , Animals , Humans , Hypoxia , Hypoxia-Inducible Factor 1, alpha Subunit/physiology , Macular Degeneration/etiology , Vascular Endothelial Growth Factor A/physiology , Vascular Endothelial Growth Factor Receptor-2/physiology , ZebrafishABSTRACT
BACKGROUND: Systemic lupus erythematosus (SLE) is a systemic autoimmune disease, which exhibits multiple B cell abnormalities including expanded populations of memory B cells and elevated levels of autoantibodies. Belimumab is a monoclonal antibody targeting the B cell cytokine BAFF (a.k.a. BLyS), approved for the treatment of SLE. METHODS: In this prospective cohort study, B cells from peripheral blood of 23 SLE patients initiating belimumab treatment and followed longitudinally for up to three years, were assessed using mass cytometry. FINDINGS: B cells decreased during the study period, with a rapid decrease of both naïve and CD11c+CD21- B cells at the first follow-up visit, followed by a continuous reduction at subsequent follow-ups. In contrast, plasma cells and switched memory B cells remained stable throughout the study. The observed immunological changes correlated with early, but not late, clinical improvements. Moreover, high baseline B cell counts were predictive of failure to attain low disease activity. In summary, our data unveiled both rapid and gradual later therapy-associated alterations of both known and unforeseen B cell phenotypes. INTERPRETATION: Our results suggest that evaluation of B cell counts might prove useful prior to initiation of belimumab treatment and that early treatment evaluation and discontinuation might underestimate delayed clinical improvements resultant of late B cell changes.
Subject(s)
Antibodies, Monoclonal, Humanized/therapeutic use , B-Cell Activating Factor/antagonists & inhibitors , B-Lymphocytes/drug effects , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Immunosuppressive Agents/therapeutic use , Lupus Erythematosus, Systemic/drug therapy , Lupus Erythematosus, Systemic/immunology , Adult , Antibodies, Monoclonal, Humanized/pharmacology , B-Lymphocyte Subsets/drug effects , B-Lymphocyte Subsets/immunology , B-Lymphocyte Subsets/metabolism , Biomarkers , Female , Humans , Immunosuppressive Agents/pharmacology , Lupus Erythematosus, Systemic/diagnosis , Lymphocyte Count , Male , Middle Aged , Severity of Illness Index , Treatment OutcomeABSTRACT
OBJECTIVE: Antibodies against posttranslationally modified proteins are a hallmark of rheumatoid arthritis (RA), but the emergence and pathogenicity of these autoantibodies are still incompletely understood. The aim of this study was to analyze the antigen specificities and mutation patterns of monoclonal antibodies (mAb) derived from RA synovial plasma cells and address the question of antigen cross-reactivity. METHODS: IgG-secreting cells were isolated from RA synovial fluid, and the variable regions of the immunoglobulins were sequenced (n = 182) and expressed in full-length mAb (n = 93) and also as germline-reverted versions. The patterns of reactivity with 53,019 citrullinated peptides and 49,211 carbamylated peptides and the potential of the mAb to promote osteoclastogenesis were investigated. RESULTS: Four unrelated anti-citrullinated protein autoantibodies (ACPAs), of which one was clonally expanded, were identified and found to be highly somatically mutated in the synovial fluid of a patient with RA. The ACPAs recognized >3,000 unique peptides modified by either citrullination or carbamylation. This highly multireactive autoantibody feature was replicated for Ig sequences derived from B cells from the peripheral blood of other RA patients. The plasma cell-derived mAb were found to target distinct amino acid motifs and partially overlapping protein targets. They also conveyed different effector functions as revealed in an osteoclast activation assay. CONCLUSION: These findings suggest that the high level of cross-reactivity among RA autoreactive B cells is the result of different antigen encounters, possibly at different sites and at different time points. This is consistent with the notion that RA is initiated in one context, such as in the mucosal organs, and thereafter targets other sites, such as the joints.
Subject(s)
Amino Acid Motifs/immunology , Anti-Citrullinated Protein Antibodies/immunology , Arthritis, Rheumatoid/immunology , Autoantigens/immunology , Immunoglobulin G/immunology , Plasma Cells/immunology , Antibodies, Monoclonal/immunology , Autoantibodies/immunology , Female , Humans , Male , Middle Aged , Protein Carbamylation , Protein Processing, Post-Translational , Synovial Fluid/cytologyABSTRACT
We aimed to evaluate in vivo effects of abatacept on phenotypes of T and B cells in the circulation of myositis patients in a sub-study of the ARTEMIS trial. Twelve patients with paired frozen PBMCs before and after 6-month abatacept treatment were included in this sub-study where mass cytometry (CyTOF) was chosen as a technology to be tested for its utility in a real-life clinical immune monitoring setting. Using CyTOF, the peripheral T cell phenotypes demonstrated considerable variation over time and between individuals precluding the identification of treatment-specific changes. We therefore conclude that studies of patient cohorts displaying wide clinical heterogeneity using mass cytometry must be relatively large in order to be suited for discovery research and immune monitoring. Still, we did find some correlations with functional muscle outcome, namely positive correlations between the ratio of CD4+ T cells and CD8+ T cells (CD4/CD8) in peripheral blood samples both at baseline and after treatment with muscle endurance improvement as assessed by the functional index-2 (FI-2) test. Our data suggest that the CD4/CD8 ratio in circulation at time of active disease may be a predictor of treatment efficacy in myositis patients.
Subject(s)
Abatacept/therapeutic use , B-Lymphocyte Subsets/drug effects , Dermatomyositis/immunology , Immunosuppressive Agents/therapeutic use , Polymyositis/drug therapy , T-Lymphocyte Subsets/drug effects , Adult , Dermatomyositis/blood , Dermatomyositis/drug therapy , Female , Humans , Male , Middle Aged , Polymyositis/blood , Polymyositis/immunologyABSTRACT
OBJECTIVE: In anti-citrullinated protein antibody positive rheumatoid arthritis (ACPA-positive RA), a particular subset of HLA-DRB1 alleles, called shared epitope (SE) alleles, is a highly influential genetic risk factor. Here, we investigated whether non-HLA single nucleotide polymorphisms (SNP), conferring low disease risk on their own, interact with SE alleles more frequently than expected by chance and if such genetic interactions influence the HLA-DRB1 SE effect concerning risk to ACPA-positive RA. METHODS: We computed the attributable proportion (AP) due to additive interaction at genome-wide level for two independent ACPA-positive RA cohorts: the Swedish epidemiological investigation of rheumatoid arthritis (EIRA) and the North American rheumatoid arthritis consortium (NARAC). Then, we tested for differences in the AP p value distributions observed for two groups of SNPs, non-associated and associated with disease. We also evaluated whether the SNPs in interaction with HLA-DRB1 were cis-eQTLs in the SE alleles context in peripheral blood mononuclear cells from patients with ACPA-positive RA (SE-eQTLs). RESULTS: We found a strong enrichment of significant interactions (AP p<0.05) between the HLA-DRB1 SE alleles and the group of SNPs associated with ACPA-positive RA in both cohorts (Kolmogorov-Smirnov test D=0.35 for EIRA and D=0.25 for NARAC, p<2.2e-16 for both). Interestingly, 564 out of 1492 SNPs in consistent interaction for both cohorts were significant SE-eQTLs. Finally, we observed that the effect size of HLA-DRB1 SE alleles for disease decreases from 5.2 to 2.5 after removal of the risk alleles of the two top interacting SNPs (rs2476601 and rs10739581). CONCLUSION: Our data demonstrate that there are massive genetic interactions between the HLA-DRB1 SE alleles and non-HLA genetic variants in ACPA-positive RA.
Subject(s)
Alleles , Arthritis, Rheumatoid/genetics , Epistasis, Genetic/genetics , Genetic Predisposition to Disease/genetics , HLA-DRB1 Chains/genetics , Anti-Citrullinated Protein Antibodies/genetics , Anti-Citrullinated Protein Antibodies/immunology , Arthritis, Rheumatoid/immunology , Cohort Studies , Epistasis, Genetic/immunology , Epitopes/genetics , Epitopes/immunology , Female , HLA-DRB1 Chains/immunology , Humans , Male , North America , Polymorphism, Single Nucleotide , Risk Factors , SwedenABSTRACT
The presence of the PTPN22 risk allele (1858T) is associated with several autoimmune diseases including rheumatoid arthritis (RA). Despite a number of studies exploring the function of PTPN22 in T cells, the exact impact of the PTPN22 risk allele on T-cell function in humans is still unclear. In this study, using RNA sequencing, we show that, upon TCR-activation, naïve human CD4+ T cells homozygous for the PTPN22 risk allele overexpress a set of genes including CFLAR and 4-1BB, which are important for cytotoxic T-cell differentiation. Moreover, the protein expression of the T-box transcription factor Eomesodermin (EOMES) was increased in T cells from healthy donors homozygous for the PTPN22 risk allele and correlated with a decreased number of naïve CD4+ T cells. There was no difference in the frequency of other CD4+ T-cell subsets (Th1, Th17, Tfh, Treg). Finally, an accumulation of EOMES+ CD4+ T cells was observed in synovial fluid of RA patients with a more pronounced production of Perforin-1 in PTPN22 risk allele carriers. Altogether, we propose a novel mechanism of action of PTPN22 risk allele through the generation of cytotoxic CD4+ T cells and identify EOMES+ CD4+ T cells as a relevant T-cell subset in RA pathogenesis.
Subject(s)
Arthritis, Rheumatoid/pathology , CD4-Positive T-Lymphocytes/immunology , Protein Tyrosine Phosphatase, Non-Receptor Type 22/genetics , T-Box Domain Proteins/metabolism , T-Lymphocytes, Cytotoxic/immunology , 4-1BB Ligand/biosynthesis , Arthritis, Rheumatoid/genetics , Base Sequence , CASP8 and FADD-Like Apoptosis Regulating Protein/biosynthesis , Cell Differentiation/immunology , Humans , Perforin/biosynthesis , Receptors, Antigen, T-Cell/immunology , Sequence Analysis, RNA , Synovial Fluid/cytology , T-Lymphocytes, Cytotoxic/cytologyABSTRACT
Non-coding SNPs in the protein tyrosine phosphatase non-receptor type 2 (PTPN2) locus have been linked with several autoimmune diseases, including rheumatoid arthritis, type I diabetes, and inflammatory bowel disease. However, the functional consequences of these SNPs are poorly characterized. Herein, we show in blood cells that SNPs in the PTPN2 locus are highly correlated with DNA methylation levels at four CpG sites downstream of PTPN2 and expression levels of the long non-coding RNA (lncRNA) LINC01882 downstream of these CpG sites. We observed that LINC01882 is mainly expressed in T cells and that anti-CD3/CD28 activated naïve CD4+ T cells downregulate the expression of LINC01882. RNA sequencing analysis of LINC01882 knockdown in Jurkat T cells, using a combination of antisense oligonucleotides and RNA interference, revealed the upregulation of the transcription factor ZEB1 and kinase MAP2K4, both involved in IL-2 regulation. Overall, our data suggests the involvement of LINC01882 in T cell activation and hints towards an auxiliary role of these non-coding SNPs in autoimmunity associated with the PTPN2 locus.
Subject(s)
Autoimmune Diseases/genetics , CD4-Positive T-Lymphocytes/physiology , CpG Islands/genetics , Protein Tyrosine Phosphatase, Non-Receptor Type 2/genetics , RNA, Long Noncoding/genetics , Autoimmunity/genetics , DNA Methylation , Gene Knockdown Techniques , Humans , Jurkat Cells , Lymphocyte Activation , MAP Kinase Kinase 4/genetics , Polymorphism, Single Nucleotide , RNA, Small Interfering/genetics , Zinc Finger E-box-Binding Homeobox 1/geneticsABSTRACT
BACKGROUND & AIMS: Alagille syndrome is a genetic disorder characterized by cholestasis, ocular abnormalities, characteristic facial features, heart defects, and vertebral malformations. Most cases are associated with mutations in JAGGED1 (JAG1), which encodes a Notch ligand, although it is not clear how these contribute to disease development. We aimed to develop a mouse model of Alagille syndrome to elucidate these mechanisms. METHODS: Mice with a missense mutation (H268Q) in Jag1 (Jag1+/Ndr mice) were outbred to a C3H/C57bl6 background to generate a mouse model for Alagille syndrome (Jag1Ndr/Ndr mice). Liver tissues were collected at different timepoints during development, analyzed by histology, and liver organoids were cultured and analyzed. We performed transcriptome analysis of Jag1Ndr/Ndr livers and livers from patients with Alagille syndrome, cross-referenced to the Human Protein Atlas, to identify commonly dysregulated pathways and biliary markers. We used species-specific transcriptome separation and ligand-receptor interaction assays to measure Notch signaling and the ability of JAG1Ndr to bind or activate Notch receptors. We studied signaling of JAG1 and JAG1Ndr via NOTCH 1, NOTCH2, and NOTCH3 and resulting gene expression patterns in parental and NOTCH1-expressing C2C12 cell lines. RESULTS: Jag1Ndr/Ndr mice had many features of Alagille syndrome, including eye, heart, and liver defects. Bile duct differentiation, morphogenesis, and function were dysregulated in newborn Jag1Ndr/Ndr mice, with aberrations in cholangiocyte polarity, but these defects improved in adult mice. Jag1Ndr/Ndr liver organoids collapsed in culture, indicating structural instability. Whole-transcriptome sequence analyses of liver tissues from mice and patients with Alagille syndrome identified dysregulated genes encoding proteins enriched at the apical side of cholangiocytes, including CFTR and SLC5A1, as well as reduced expression of IGF1. Exposure of Notch-expressing cells to JAG1Ndr, compared with JAG1, led to hypomorphic Notch signaling, based on transcriptome analysis. JAG1-expressing cells, but not JAG1Ndr-expressing cells, bound soluble Notch1 extracellular domain, quantified by flow cytometry. However, JAG1 and JAG1Ndr cells each bound NOTCH2, and signaling from NOTCH2 signaling was reduced but not completely inhibited, in response to JAG1Ndr compared with JAG1. CONCLUSIONS: In mice, expression of a missense mutant of Jag1 (Jag1Ndr) disrupts bile duct development and recapitulates Alagille syndrome phenotypes in heart, eye, and craniofacial dysmorphology. JAG1Ndr does not bind NOTCH1, but binds NOTCH2, and elicits hypomorphic signaling. This mouse model can be used to study other features of Alagille syndrome and organ development.
Subject(s)
Alagille Syndrome/genetics , Jagged-1 Protein/genetics , Mutation, Missense , Alagille Syndrome/metabolism , Alagille Syndrome/pathology , Animals , Bile Ducts, Intrahepatic/metabolism , Bile Ducts, Intrahepatic/pathology , Cell Differentiation , Coculture Techniques , Disease Models, Animal , Female , Gene Expression Profiling/methods , Gene Expression Regulation, Developmental , Genetic Predisposition to Disease , HEK293 Cells , Humans , Jagged-1 Protein/metabolism , Male , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Transgenic , Morphogenesis , Organoids , Phenotype , Receptor, Notch2/genetics , Receptor, Notch2/metabolism , Signal Transduction , TransfectionABSTRACT
This manuscript is a companion paper to Amara et al. [1]. Data shown here include detailed clinical characteristics from anonymized patients, the Ig subclass data generated from B cells sorted from four individual patients, tables detailing variable gene region sequences from sorted cells linked to the patient information and the sequence yields from individual patients. Furthermore a URL link to the RNAseq datasets submitted to GEO is included.
ABSTRACT
The clinical efficacy of B cell targeting therapies highlights the pathogenic potential of B cells in inflammatory diseases. Expression of Fc Receptor like 4 (FcRL4) identifies a memory B cell subset, which is enriched in the joints of patients with rheumatoid arthritis (RA) and in mucosa-associated lymphoid tissue. The high level of RANKL production by this B cell subset indicates a unique pathogenic role. In addition, recent work has identified a role for FcRL4 as an IgA receptor, suggesting a potential function in mucosal immunity. Here, the contribution of FcRL4+ B cells to the specific autoimmune response in the joints of patients with RA was investigated. Single FcRL4+ and FcRL4- B cells were sorted from synovial fluid and tissue from RA patients and their immunoglobulin genes characterized. Levels of hypermutation in the variable regions in both populations were largely consistent with memory B cells selected by an antigen- and T cell-dependent process. Recombinant antibodies were generated based on the IgH and IgL variable region sequences and investigated for antigen specificity. A significantly larger proportion of the recombinant antibodies generated from individual synovial FcRL4+ B cells showed reactivity towards citrullinated autoantigens. Furthermore, both in analyses based on heavy chain sequences and flow cytometric detection, FcRL4+ B cells have significantly increased usage of the IgA isotype. Their low level of expression of immunoglobulin and plasma cell differentiation genes does not suggest current antibody secretion. We conclude that these activated B cells are a component of the local autoimmune response, and through their RANKL expression, can contribute to joint destruction. Furthermore, their expression of FcRL4 and their enrichment in the IgA isotype points towards a potential role for these cells in the link between mucosal and joint inflammation.
Subject(s)
Arthritis, Rheumatoid/genetics , Arthritis, Rheumatoid/immunology , Autoimmunity/genetics , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Gene Expression , Receptors, Fc/genetics , Aged , Arthritis, Rheumatoid/diagnosis , Arthritis, Rheumatoid/metabolism , Autoantigens/immunology , Biomarkers , Female , Humans , Immunoglobulin Isotypes/genetics , Immunoglobulin Isotypes/immunology , Immunoglobulins/genetics , Immunoglobulins/immunology , Lymphocyte Activation/immunology , Male , Middle Aged , Mutation , Synovial Fluid/immunology , Synovial Membrane/immunology , TranscriptomeABSTRACT
OBJECTIVES: Belimumab is the first biologic drug approved for Systemic Lupus Erythematosus (SLE). Here, we aimed to investigate the effects of belimumab on clinical and serologic outcomes, and sought to identify predictors of treatment response in three Swedish real-life settings. METHODS: Fifty-eight patients were enrolled at initiation of belimumab and followed longitudinally for up to 53months. Surveillance outcomes included the SLE Disease Activity Index 2000 (SLEDAI-2K), 100mm Visual Analogue Scales for Physician's Global Assessment (PGA), fatigue, pain and general health, and the Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index (SDI). Assessment of treatment response included the SLE responder index (SRI). B lymphocyte stimulator (BLyS) levels were determined using ELISA. RESULTS: SLEDAI-2K (median baseline score: 8.0; IQR: 4.0-13.8), PGA and corticosteroid use decreased during therapy, and patients reported improvements on fatigue, pain, and general health (p<0.0001 for all). SDI scores remained stable (p=0.08). Patients with baseline SDI scores >1 showed decreased probability and prolonged time to attain SRI response (HR: 0.449; 95% CI: 0.208-0.967), as did current smokers compared with non-smokers (HR: 0.103; 95% CI: 0.025-0.427). In contrast, baseline BLyS levels ≥1.2ng/mL predicted increased probability and shorter time to attain SRI response (HR: 2.566; 95% CI: 1.222-5.387). CONCLUSIONS: Disease activity and corticosteroid usage decreased, patient-reported outcomes improved, and no significant organ damage was accrued during follow-up. Smoking and organ damage predicted reduced treatment efficacy. These findings might contribute to a better selection of patients who are likely to benefit from belimumab.
Subject(s)
Antibodies, Monoclonal, Humanized/pharmacology , Lupus Erythematosus, Systemic/drug therapy , Severity of Illness Index , Smoking/adverse effects , Adult , Female , Humans , Lupus Erythematosus, Systemic/immunology , Male , Middle Aged , Treatment OutcomeABSTRACT
Cellular heterogeneity can emerge from the expression of only one parental allele. However, it has remained controversial whether, or to what degree, random monoallelic expression of autosomal genes (aRME) is mitotically inherited (clonal) or stochastic (dynamic) in somatic cells, particularly in vivo. Here we used allele-sensitive single-cell RNA-seq on clonal primary mouse fibroblasts and freshly isolated human CD8+ T cells to dissect clonal and dynamic monoallelic expression patterns. Dynamic aRME affected a considerable portion of the cells' transcriptomes, with levels dependent on the cells' transcriptional activity. Notably, clonal aRME was detected, but it was surprisingly scarce (<1% of genes) and mainly affected the most weakly expressed genes. Consequently, the overwhelming majority of aRME occurs transiently within individual cells, and patterns of aRME are thus primarily scattered throughout somatic cell populations rather than, as previously hypothesized, confined to patches of clonally related cells.