Recent technological advances have enabled massively parallel chromatin profiling with scATAC-seq (single-cell assay for transposase accessible chromatin by sequencing). Here we present ATAC with ...select antigen profiling by sequencing (ASAP-seq), a tool to simultaneously profile accessible chromatin and protein levels. Our approach pairs sparse scATAC-seq data with robust detection of hundreds of cell surface and intracellular protein markers and optional capture of mitochondrial DNA for clonal tracking, capturing three distinct modalities in single cells. ASAP-seq uses a bridging approach that repurposes antibody:oligonucleotide conjugates designed for existing technologies that pair protein measurements with single-cell RNA sequencing. Together with DOGMA-seq, an adaptation of CITE-seq (cellular indexing of transcriptomes and epitopes by sequencing) for measuring gene activity across the central dogma of gene regulation, we demonstrate the utility of systematic multi-omic profiling by revealing coordinated and distinct changes in chromatin, RNA and surface proteins during native hematopoietic differentiation and peripheral blood mononuclear cell stimulation and as a combinatorial decoder and reporter of multiplexed perturbations in primary T cells.
In the middle of the 20th century, biologists focused on investigating the mechanism of gene regulation and signal transduction in cells, which led to the concept that metabolites were products of ...gene expression and signal transduction pathways. In the 1920s, the importance of cellular metabolism was shown in the Warburg effect, in which cancer cells are characterized by a mitochondrial defect that shifts towards aerobic glycolysis. Recently, it is accepted that each organ and cell subset needs specific metabolic conditions and metabolic regulatory systems.
Immunometabolism is a relatively new field of metabolism studies. The immune system consists of various cell subsets that have unique requirements and functions. The metabolic reprogramming in each immune cell causes different effects on different cell subsets. For example, resting lymphocytes generate energy through oxidative phosphorylation (OXPHOS) and fatty acid oxidation (FAO), whereas activated lymphocytes rapidly shift to the glycolytic pathway. A detailed understanding of metabolic regulation has progressed rapidly, especially in T cells during their differentiation from naïve to effector T cells.
Metabolism is now considered to play a key role in autoimmune diseases. Metabolic changes in autoimmune diseases might be due to inflammation as well as being involved in autoimmune pathogenesis. Systemic lupus erythematosus (SLE) is an autoimmune disease with heterogenous clinical presentations whose precise pathophysiological mechanism is largely unknown. In this report, we review the altered metabolism in SLE and discuss the potential of metabolomics for accelerating the discovery of novel cellular autoimmune therapies and novel disease biomarkers.
Abstract
Myasthenia gravis (MG) is a neurological disease caused by autoantibodies against neuromuscular-associated proteins. While MG frequently develops in thymoma patients, the etiologic factors ...for MG are not well understood. Here, by constructing a comprehensive atlas of thymoma using bulk and single-cell RNA-sequencing, we identify ectopic expression of neuromuscular molecules in MG-type thymoma. These molecules are found within a distinct subpopulation of medullary thymic epithelial cells (mTECs), which we name neuromuscular mTECs (nmTECs). MG-thymoma also exhibits microenvironments dedicated to autoantibody production, including ectopic germinal center formation, T follicular helper cell accumulation, and type 2 conventional dendritic cell migration. Cell–cell interaction analysis also predicts the interaction between nmTECs and T/B cells via
CXCL12
-
CXCR4
. The enrichment of nmTECs presenting neuromuscular molecules within MG-thymoma is further confirmed immunohistochemically and by cellular composition estimation from the MG-thymoma transcriptome. Altogether, this study suggests that nmTECs have a significant function in MG pathogenesis via ectopic expression of neuromuscular molecules.
Abstract
Background
Behçet’s syndrome (BS) is an immune-mediated disease characterized by recurrent oral ulcers, genital ulcers, uveitis, and skin symptoms. HLA-B51, as well as other genetic ...polymorphisms, has been reported to be associated with BS; however, the pathogenesis of BS and its relationship to genetic risk factors still remain unclear. To address these points, we performed immunophenotyping and transcriptome analysis of immune cells from BS patients and healthy donors.
Methods
ImmuNexUT is a comprehensive database consisting of RNA sequencing data and eQTL database of immune cell subsets from patients with immune-mediated diseases and healthy donors, and flow cytometry data and transcriptome data from 23 BS patients and 28 healthy donors from the ImmuNexUT study were utilized for this study. Differential gene expression analysis and weighted gene co-expression network analysis (WGCNA) were performed to identify genes associated with BS and clinical features of BS. eQTL database was used to assess the relationship between genetic risk factors of BS with those genes.
Results
The frequency of Th17 cells was increased in BS patients, and transcriptome analysis of Th17 cells suggested the activation of the NFκB pathway in Th17 cells of BS patients. Next, WGCNA was used to group genes into modules with similar expression patterns in each subset. Modules of antigen-presenting cells were associated with BS, and pathway analysis suggested the activation of antigen-presenting cells of BS patients. Further examination of genes in BS-associated modules indicated that the expression of
YBX3
, a member of a plasmacytoid dendritic cell (pDC) gene module associated with BS, is influenced by a BS risk polymorphism, rs2617170, in pDCs, suggesting that
YBX3
may be a key molecule connecting genetic risk factors of BS with disease pathogenesis. Furthermore, pathway analysis of modules associated with HLA-B51 indicated that the association of IL-17-associated pathways in memory CD8
+
T cells with HLA-B51; therefore, IL-17-producing CD8
+
T cells, Tc17 cells, may play a critical role in BS.
Conclusions
Various cells including CD4
+
T cells, CD8
+
T cells, and antigen-presenting cells are important in the pathogenesis of BS. Tc17 cells and
YBX3
may be potential therapeutic targets in BS.
To investigate metabolite alterations in the plasma of SLE patients to identify novel biomarkers and provide insight into SLE pathogenesis.
Patients with SLE (n = 41, discovery cohort and n = 37, ...replication cohort), healthy controls (n = 30 and n = 29) and patients with RA (n = 19, disease control) were recruited. Metabolic profiles of the plasma samples were analysed using liquid chromatography-time-of-flight mass spectrometry and capillary electrophoresis-time-of-flight mass spectrometry. Transcriptome data was analysed using RNA-sequencing for 18 immune cell subsets. The importance of histidine (His) in plasmablast differentiation was investigated by using mouse splenic B cells.
We demonstrate that a specific amino acid combination including His can effectively distinguish between SLE patients and healthy controls. Random forest and partial least squares-discriminant analysis identified His as an effective classifier for SLE patients. A decrease in His plasma levels correlated with damage accrual independent of prednisolone dosage and type I IFN signature. The oxidative phosphorylation signature in plasmablasts negatively correlated with His levels. We also showed that plasmablast differentiation induced by innate immune signals was dependent on His.
Plasma His levels are a potential biomarker for SLE patients and are associated with damage accrual. Our data suggest the importance of His as a pathogenic metabolite in SLE pathogenesis.
Almost 160 years after the discovery of mitochondria, they are known for their production of energy and are called "the powerhouse of the cell". Recently, immune-metabolism has been revealed as a key ...factor controlling immune cell proliferation and differentiation. Resting lymphocytes generate energy through oxidative phosphorylation and fatty acid oxidation, whereas activated lymphocytes rapidly shift to glycolysis. Oxidative phosphorylation (OXPHOS) as well as mitochondrial reactive oxygen species (mtROS) generated through the electron transport chain (ETC) are involved in many immune cell functions. Moreover, mitochondria are dynamic organelles that can provide immunogenic molecules, such as mitochondrial DNA (mtDNA) resulting in innate immune system activation. Here, we describe the role of mitochondria in immune system regulation, highlighting metabolism-dependent and other immunogenic aspects.
Previous gene expression analyses seeking genes specific to antineutrophil cytoplasmic antibody-associated vasculitis (AAV) have been limited due to crude cell separation and the use of microarrays. ...This study aims to identify AAV-specific gene expression profiles in a way that overcomes those limitations.
Blood samples were collected from 26 AAV patients and 28 healthy controls (HCs). Neutrophils were isolated by negative selection, whereas 19 subsets of peripheral blood mononuclear cells were sorted by fluorescence assisted cell sorting. RNA-sequencing was then conducted for each sample, and iterative weighted gene correlation network analysis (iterativeWGCNA) and random forest were consecutively applied to identify the most influential gene module in distinguishing AAV from HCs. Correlations of the identified module with clinical parameters were evaluated, and the biological role was assessed with hub gene identification and pathway analysis. Particularly, the module's association with neutrophil extracellular trap formation, NETosis, was analyzed. Finally, the module's overlap with GWAS-identified autoimmune disease genes (GADGs) was assessed for validation.
A neutrophil module (Neu_M20) was ranked top in the random forest analysis among 255 modules created by iterativeWGCNA. Neu_M20 correlated with disease activity and neutrophil counts but not with the presence of antineutrophil cytoplasmic antibody. The module comprised pro-inflammatory genes, including those related to NETosis, supported by experimental evidence. The genes in the module significantly overlapped GADGs.
We identified the distinct group of pro-inflammatory genes in neutrophils, which characterize AAV. Further investigations are warranted to confirm our findings as they could serve as novel therapeutic targets.
•Expression profile of a gene module in neutrophils characterizes AAV.•The module eigengene correlates with disease activity and neutrophil counts.•The module comprises pro-inflammatory genes, including those related to NETosis.•Genes in the module overlap autoimmune disease genes identified by GWAS.
Systemic lupus erythematosus (SLE), which was recognized as a defined clinical entity more than 100 years ago, is an archetype for systemic autoimmune diseases. The 10-year survival of SLE patients ...has shown dramatic improvement during the last half-century. However, SLE patients receiving long-term prednisone therapy are at high risk of morbidity due to organ damage. Identification of key immune pathways is mandatory to develop a suitable therapy and to stratify patients based on their responses to therapy. Recently developed transcriptome and omic analyses have revealed a number of immune pathways associated with systemic autoimmunity. In addition to type I interferon, plasmablast and neutrophil signatures demonstrate associations with the SLE phenotype. Systematic investigations of these findings enable us to understand and stratify SLE according to the clinical and immunological features.
Consecutive mRNA vaccinations against SARS-CoV-2 reinforced both innate and adaptive immune responses. However, it remains unclear whether the enhanced innate immune responses are mediated by ...epigenetic regulation and, if so, whether these effects persist. Using mass cytometry, RNA-Seq, and ATAC-Seq, we show that BNT162b2 mRNA vaccination upregulated antiviral and IFN-stimulated gene expression in monocytes with greater effects after the second vaccination than those after the first vaccination. Transcription factor-binding motif analysis also revealed enriched IFN regulatory factors and PU.1 motifs in accessible chromatin regions. Importantly, although consecutive BNT162b2 mRNA vaccinations boosted innate immune responses and caused epigenetic changes in isolated monocytes, we show that these effects occurred only transiently and disappeared 4 weeks after the second vaccination. Furthermore, single-cell RNA-Seq analysis revealed that a similar gene signature was impaired in the monocytes of unvaccinated patients with COVID-19 with acute respiratory distress syndrome. These results reinforce the importance of the innate immune response in the determination of COVID-19 severity but indicate that, unlike adaptive immunity, innate immunity is not unexpectedly sustained even after consecutive vaccination. This study, which focuses on innate immune memory, may provide novel insights into the vaccine development against infectious diseases.
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