Thioredoxin is a metabolic rheostat controlling regulatory B cells.

Metabolic programming is important for B cell fate, but the bioenergetic requirement for regulatory B (Breg) cell differentiation and function is unknown. Here we show that Breg cell differentiation, unlike non-Breg cells, relies on mitochondrial electron transport and homeostatic levels of reactive oxygen species (ROS). Single-cell RNA sequencing analysis revealed that TXN, encoding the metabolic redox protein thioredoxin (Trx), is highly expressed by Breg cells, unlike Trx inhibitor TXNIP which was downregulated. Pharmacological inhibition or gene silencing of TXN resulted in mitochondrial membrane depolarization and increased ROS levels, selectively suppressing Breg cell differentiation and function while favoring pro-inflammatory B cell differentiation. Patients with systemic lupus erythematosus (SLE), characterized by Breg cell deficiencies, present with B cell mitochondrial membrane depolarization, elevated ROS and fewer Trx+ B cells. Exogenous Trx stimulation restored Breg cells and mitochondrial membrane polarization in SLE B cells to healthy B cell levels, indicating Trx insufficiency underlies Breg cell impairment in patients with SLE.

Differential regulation of IL-17A and IL-17F via STAT5 contributes to psoriatic disease.

BACKGROUND: IL-17A plays a pivotal pathogenic role in several immune-mediated inflammatory diseases. Despite sharing 50% sequence homology with IL-17A, the role of IL-17F remains less clear. Clinical findings suggest that dual inhibition of IL-17A and IL-17F in psoriatic disease is more efficacious than IL-17A inhibition alone, positing a pathogenic role for IL-17F. OBJECTIVE: We characterized the regulation of IL-17A and IL-17F in psoriatic disease. METHODS: Using both in vitro systems and lesional skin tissue from patients, we interrogated the chromosomal, transcriptional, and protein expression landscape of IL-17A+ and IL-17F+ TH17 cells. Alongside established assays such as single-cell RNA sequencing, we developed a novel cytokine-capture technique that was combined with chromatin immunoprecipitation sequencing and RNA sequencing. RESULTS: We confirm a preferential elevation of IL-17F over IL-17A in psoriatic disease and show that expression of each isoform predominantly occurs in distinct cell populations. The expression of both IL-17A and IL-17F exhibited a high degree of plasticity, with the balance between the 2 isoforms influenced by proinflammatory signaling and by anti-inflammatory drugs such as methylprednisolone. This plasticity was reflected in a broad H3K4me3 region at the IL17A-F locus, while opposing effects of STAT5/IL-2 signaling were observed for each of the 2 genes. Functionally, higher IL17F expression was linked to greater cell proliferation. CONCLUSION: There are key differences in the regulation of IL-17A and IL-17F in psoriatic disease, leading to distinct inflammatory cell populations. As such, we propose that both IL-17A and IL-17F neutralization may be required to maximally inhibit IL-17-driven pathology.

DNA methylation analysis identifies key transcription factors involved in mesenchymal stem cell osteogenic differentiation.

BACKGROUND: Knowledge about regulating transcription factors (TFs) for osteoblastogenesis from mesenchymal stem cells (MSCs) is limited. Therefore, we investigated the relationship between genomic regions subject to DNA-methylation changes during osteoblastogenesis and the TFs known to directly interact with these regulatory regions. RESULTS: The genome-wide DNA-methylation signature of MSCs differentiated to osteoblasts and adipocytes was determined using the Illumina HumanMethylation450 BeadChip array. During adipogenesis no CpGs passed our test for significant methylation changes. Oppositely, during osteoblastogenesis we identified 2462 differently significantly methylated CpGs (adj. p < 0.05). These resided outside of CpGs islands and were significantly enriched in enhancer regions. We confirmed the correlation between DNA-methylation and gene expression. Accordingly, we developed a bioinformatic tool to analyse differentially methylated regions and the TFs interacting with them. By overlaying our osteoblastogenesis differentially methylated regions with ENCODE TF ChIP-seq data we obtained a set of candidate TFs associated to DNA-methylation changes. Among them, ZEB1 TF was highly related with DNA-methylation. Using RNA interference, we confirmed that ZEB1, and ZEB2, played a key role in adipogenesis and osteoblastogenesis processes. For clinical relevance, ZEB1 mRNA expression in human bone samples was evaluated. This expression positively correlated with weight, body mass index, and PPARγ expression. CONCLUSIONS: In this work we describe an osteoblastogenesis-associated DNA-methylation profile and, using these data, validate a novel computational tool to identify key TFs associated to age-related disease processes. By means of this tool we identified and confirmed ZEB TFs as mediators involved in the MSCs differentiation to osteoblasts and adipocytes, and obesity-related bone adiposity.

microRNA-seq of cartilage reveals an overabundance of miR-140-3p which contains functional isomiRs.

miR-140 is selectively expressed in cartilage. Deletion of the entire Mir140 locus in mice results in growth retardation and early-onset osteoarthritis-like pathology; however, the relative contribution of miR-140-5p or miR-140-3p to the phenotype remains to be determined. An unbiased small RNA sequencing approach identified miR-140-3p as significantly more abundant (>10-fold) than miR-140-5p in human cartilage. Analysis of these data identified multiple miR-140-3p isomiRs differing from the miRBase annotation at both the 5' and 3' end, with >99% having one of two seed sequences (5' bases 2-8). Canonical (miR-140-3p.2) and shifted (miR-140-3p.1) seed isomiRs were overexpressed in chondrocytes and transcriptomics performed to identify targets. miR-140-3p.1 and miR-140-3p.2 significantly down-regulated 694 and 238 genes, respectively, of which only 162 genes were commonly down-regulated. IsomiR targets were validated using 3'UTR luciferase assays. miR-140-3p.1 targets were enriched within up-regulated genes in rib chondrocytes of Mir140-null mice and within down-regulated genes during human chondrogenesis. Finally, through imputing the expression of miR-140 from the expression of the host gene WWP2 in 124 previously published data sets, an inverse correlation with miR-140-3p.1 predicted targets was identified. Together these data suggest the novel seed containing isomiR miR-140-3p.1 is more functional than original consensus miR-140-3p seed containing isomiR.

Regulation of microRNA-221, -222, -21 and -27 in articular cartilage subjected to abnormal compressive forces.

KEY POINTS: microRNAs (miRs) are small non-coding molecules that regulate post-transcriptional target gene expression. miRs are involved in regulating cellular activities in response to mechanical loading in all physiological systems, although it is largely unknown whether this response differs with increasing magnitudes of load. miR-221, miR-222, miR-21-5p and miR-27a-5p were significantly increased in ex vivo cartilage explants subjected to increasing load magnitude and in in vivo joint cartilage exposed to abnormal loading. TIMP3 and CPEB3 are putative miR targets in chondrocytes Identification of mechanically regulated miRs that have potential to impact on tissue homeostasis provides a mechanism by which load-induced tissue behaviour is regulated, in both health and pathology, in all physiological systems. ABSTRACT: MicroRNAs (miRs) are small non-coding molecules that regulate post-transcriptional target gene expression and are involved in mechano-regulation of cellular activities in all physiological systems. It is unknown whether such epigenetic mechanisms are regulated in response to increasing magnitudes of load. The present study investigated mechano-regulation of miRs in articular cartilage subjected to 'physiological' and 'non-physiological' compressive loads in vitro as a model system and validated findings in an in vivo model of abnormal joint loading. Bovine full-depth articular cartilage explants were loaded to 2.5 MPa (physiological) or 7 MPa (non-physiological) (1 Hz, 15 min) and mechanically-regulated miRs identified using next generation sequencing and verified using a quantitative PCR. Downstream targets were verified using miR-specific mimics or inhibitors in conjunction with 3'-UTR luciferase activity assays. A subset of miRs were mechanically-regulated in ex vivo cartilage explants and in vivo joint cartilage. miR-221, miR-222, miR-21-5p and miR-27a-5p were increased and miR-483 levels decreased with increasing load magnitude. Tissue inhibitor of metalloproteinase 3 (TIMP3) and cytoplasmic polyadenylation element binding protein 3 (CPEB3) were identified as putative downstream targets. Our data confirm miR-221 and -222 mechano-regulation and demonstrates novel mechano-regulation of miR-21-5p and miR-27a-5p in ex vivo and in vivo cartilage loading models. TIMP3 and CPEB3 are putative miR targets in chondrocytes. Identification of specific miRs that are regulated by increasing load magnitude, as well as their potential to impact on tissue homeostasis, has direct relevance to other mechano-sensitive physiological systems and provides a mechanism by which load-induced tissue behaviour is regulated, in both health and pathology.

Lymphocyte DNA methylation mediates genetic risk at shared immune-mediated disease loci.

BACKGROUND: Defining regulatory mechanisms through which noncoding risk variants influence the cell-mediated pathogenesis of immune-mediated disease (IMD) has emerged as a priority in the post-genome-wide association study era. OBJECTIVES: With a focus on rheumatoid arthritis, we sought new insight into genetic mechanisms of adaptive immune dysregulation to help prioritize molecular pathways for targeting in this and related immune pathologies. METHODS: Whole-genome methylation and transcriptional data from isolated CD4+ T cells and B cells of more than 100 genotyped and phenotyped patients with inflammatory arthritis, all of whom were naive to immunomodulatory treatments, were obtained. Analysis integrated these comprehensive data with genome-wide association study findings across IMDs and other publicly available resources. RESULTS: We provide strong evidence that disease-associated DNA variants regulate cis-CpG methylation in CD4+ T and/or B cells at 37% RA loci. Using paired, cell-specific transcriptomic data and causal inference testing, we identify examples where site-specific DNA methylation in turn mediates gene expression, including FCRL3 in both cell types and ORMDL3/GSDMB, IL6ST/ANKRD55, and JAZF1 in CD4+ T cells. A number of genes regulated in this way highlight mechanisms common to RA and other IMDs including multiple sclerosis and asthma, in turn distinguishing them from osteoarthritis, a primarily degenerative disease. Finally, we corroborate the observed effects experimentally. CONCLUSIONS: Our observations highlight important mechanisms of genetic risk in RA and the wider context of immune dysregulation. They confirm the utility of DNA methylation profiling as a tool for causal gene prioritization and, potentially, therapeutic targeting in complex IMD.

The long non-coding RNA ROCR contributes to SOX9 expression and chondrogenic differentiation of human mesenchymal stem cells.

Long non-coding RNAs (lncRNAs) are expressed in a highly tissue-specific manner and function in various aspects of cell biology, often as key regulators of gene expression. In this study, we established a role for lncRNAs in chondrocyte differentiation. Using RNA sequencing we identified a human articular chondrocyte repertoire of lncRNAs from normal hip cartilage donated by neck of femur fracture patients. Of particular interest are lncRNAs upstream of the master chondrocyte transcription factor SOX9 locus. SOX9 is an HMG-box transcription factor that plays an essential role in chondrocyte development by directing the expression of chondrocyte-specific genes. Two of these lncRNAs are upregulated during chondrogenic differentiation of mesenchymal stem cells (MSCs). Depletion of one of these lncRNAs, LOC102723505, which we termed ROCR (regulator of chondrogenesis RNA), by RNA interference disrupted MSC chondrogenesis, concomitant with reduced cartilage-specific gene expression and incomplete matrix component production, indicating an important role in chondrocyte biology. Specifically, SOX9 induction was significantly ablated in the absence of ROCR, and overexpression of SOX9 rescued the differentiation of MSCs into chondrocytes. Our work sheds further light on chondrocyte-specific SOX9 expression and highlights a novel method of chondrocyte gene regulation involving a lncRNA.

RNASeq_similarity_matrix: visually identify sample mix-ups in RNASeq data using a 'genomic' sequence similarity matrix.

MOTIVATION: Mistakes in linking a patient's biological samples with their phenotype data can confound RNA-Seq studies. The current method for avoiding such sample mixups is to test for inconsistencies between biological data and known phenotype data such as sex. However, in DNA studies a common QC step is to check for unexpected relatedness between samples. Here, we extend this method to RNA-Seq, which allows the detection of duplicated samples without relying on identifying inconsistencies with phenotype data. SUMMARY: We present RNASeq_similarity_matrix: an automated tool to generate a sequence similarity matrix from RNA-Seq data, which can be used to visually identify sample mix-ups. This is particularly useful when a study contains multiple samples from the same individual, but can also detect contamination in studies with only one sample per individual. AVAILABILITY: RNASeq_similarity_matrix has been made available as a documented GPL licensed Docker image on www.github.com/nicokist/RNASeq_similarity_matrix. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Isolation and next generation sequencing of archival formalin-fixed DNA.

DNA from archived organs is presumed unsuitable for genomic studies because of excessive formalin-fixation. As next generation sequencing (NGS) requires short DNA fragments, and Uracil-N-glycosylase (UNG) can be used to overcome deamination, there has been renewed interest in the possibility of genomic studies using these collections. We describe a novel method of DNA extraction capable of providing PCR amplicons of at least 400 bp length from such excessively formalin-fixed human tissues. When compared with a leading commercial formalin-fixed DNA extraction kit, our method produced greater yields of DNA and reduced sequence variations. Analysis of PCR products using bacterial sub-cloning and Sanger sequencing from UNG-treated DNA unexpectedly revealed increased sequence variations, compared with untreated samples. Finally, whole exome NGS was performed on a myocardial sample fixed in formalin for 2 years and compared with lymphocyte-derived DNA (as a gold standard) from the same patient. Despite the reduction in the number and quality of reads in the formalin-fixed DNA, we were able to show that bioinformatic processing by joint calling and variant quality score recalibration (VQSR) increased the sensitivity four-fold to 56% and doubled specificity to 68% when compared with a standard hard-filtering approach. Thus, high-quality DNA can be extracted from excessively formalin-fixed tissues and bioinformatic processing can optimise sensitivity and specificity of results. Sequencing of several sub-cloned amplicons is an important methodological step in assessing DNA quality.

Predicting drug-free remission in rheumatoid arthritis: A prospective interventional cohort study.

BACKGROUND: Many patients with rheumatoid arthritis (RA) achieve disease remission with modern treatment strategies. However, having achieved this state, there are no tests that predict when withdrawal of therapy will result in drug-free remission rather than flare. We aimed to identify predictors of drug-free remission in RA. METHODS: The Biomarkers of Remission in Rheumatoid Arthritis (BioRRA) Study was a unique, prospective, interventional cohort study of complete and abrupt cessation of conventional synthetic disease-modifying anti-rheumatic drugs (DMARDs). Patients with RA of at least 12 months duration and in clinical and ultrasound remission discontinued DMARDs and were monitored for six months. The primary outcome was time-to-flare, defined as disease activity score in 28 joints with C-reactive protein (DAS28-CRP) ≥ 2.4. Baseline clinical and ultrasound measures, circulating inflammatory biomarkers, and peripheral CD4+ T cell gene expression were assessed for their ability to predict time-to-flare and flare/remission status by Cox regression and receiver-operating characteristic (ROC) analysis respectively. RESULTS: 23/44 (52%) eligible patients experienced an arthritis flare after a median (IQR) of 48 (31.5-86.5) days following DMARD cessation. A composite score incorporating five baseline variables (three transcripts [FAM102B, ENSG00000228010, ENSG00000227070], one cytokine [interleukin-27], one clinical [Boolean remission]) differentiated future flare from drug-free remission with an area under the ROC curve of 0.96 (95% CI 0.91-1.00), sensitivity 0.91 (0.78-1.00) and specificity 0.95 (0.84-1.00). CONCLUSION: We provide proof-of-concept evidence for predictors of drug-free remission in RA. If validated, these biomarkers could help to personalize immunosuppressant withdrawal: a therapy paradigm shift with ensuing patient and economic benefits.

Expression of STAT3-regulated genes in circulating CD4+ T cells discriminates rheumatoid arthritis independently of clinical parameters in early arthritis.

OBJECTIVES: Dysregulated signal transduction and activator of transcription-3 (STAT3) signalling in CD4+ T cells has been proposed as an early pathophysiological event in RA. We sought further evidence for this observation, and to determine its clinical relevance. METHODS: Microarray technology was used to measure gene expression in purified peripheral blood CD4+ T cells from treatment-naïve RA patients and disease controls newly recruited from an early arthritis clinic. Analysis focused on 12 previously proposed transcripts, and concurrent STAT3 pathway activation was determined in the same cells by flow cytometry. A pooled analysis of previous and current gene expression findings incorporated detailed clinical parameters and employed multivariate analysis. RESULTS: In an independent cohort of 161 patients, expression of 11 of 12 proposed signature genes differed significantly between RA patients and controls, robustly validating the earlier findings. Differential regulation was most pronounced for the STAT3 target genes PIM1, BCL3 and SOCS3 (>1.3-fold difference; P < 0.005), each of whose expression correlated strongly with paired intracellular phospho-STAT3. In a meta-analysis of 279 patients the same three genes accounted for the majority of the signature's ability to discriminate RA patients, which was found to be independent of age, joint involvement or acute phase response. CONCLUSION: The STAT3-mediated dysregulation of BCL3, SOCS3 and PIM1 in circulating CD4+ T cells is a discriminatory feature of early RA that occurs independently of acute phase response. The mechanistic and functional implications of this observation at a cellular level warrant clarification.

Cytokine-induced MMP13 Expression in Human Chondrocytes Is Dependent on Activating Transcription Factor 3 (ATF3) Regulation.

Irreversible breakdown of cartilage extracellular matrix (ECM) by the collagenase matrix metalloproteinase 13 (MMP13) represents a key event in osteoarthritis (OA) progression. Although inflammation is most commonly associated with inflammatory joint diseases, it also occurs in OA and is thus relevant to the prevalent tissue destruction. Here, inflammation generates a cFOS AP-1 early response that indirectly affects MMP13 gene expression. To ascertain a more direct effect on prolonged MMP13 production we examined the potential molecular events occurring between the rapid, transient expression of cFOS and the subsequent MMP13 induction. Importantly, we show MMP13 mRNA expression is mirrored by nascent hnRNA transcription. Employing ChIP assays, cFOS recruitment to the MMP13 promoter occurs at an early stage prior to gene transcription and that recruitment of transcriptional initiation markers also correlated with MMP13 expression. Moreover, protein synthesis inhibition following early FOS expression resulted in a significant decrease in MMP13 expression thus indicating a role for different regulatory factors modulating expression of the gene. Subsequent mRNA transcriptome analyses highlighted several genes induced soon after FOS that could contribute to MMP13 expression. Specific small interfering RNA-mediated silencing highlighted that ATF3 was as highly selective for MMP13 as cFOS. Moreover, ATF3 expression was AP-1(cFOS/cJUN)-dependent and expression levels were maintained after the early transient cFOS response. Furthermore, ATF3 bound the proximal MMP13 AP-1 motif in stimulated chondrocytes at time points that no longer supported binding of FOS Consequently, these findings support roles for both cFOS (indirect) and ATF3 (direct) in effecting MMP13 transcription in human chondrocytes.

The first international workshop on the epigenetics of osteoarthritis.

Osteoarthritis (OA) is a major clinical problem across the world, in part due to the lack of disease-modifying drugs resulting, to a significant degree, from our incomplete understanding of the underlying molecular mechanisms of the disease. Emerging evidence points to a role of epigenetics in the pathogenesis of OA, but research in this area is still in its early stages. In order to summarize current knowledge and to facilitate the potential coordination of future research activities, the first international workshop on the epigenetics of OA was held in Amsterdam in October 2015. Recent findings on DNA methylation and hydroxymethylation, histone modifications, noncoding RNAs, and other epigenetic mechanisms were presented and discussed. The workshop demonstrated the advantage of bringing together those working in this nascent field and highlights from the event are summarized in this report in the form of summaries from invited speakers and organizers.

Expression analysis of the osteoarthritis genetic susceptibility locus mapping to an intron of the MCF2L gene and marked by the polymorphism rs11842874.

BACKGROUND: Osteoarthritis (OA) is a painful, debilitating disease characterised by loss of articular cartilage with concurrent changes in other tissues of the synovial joint. Genetic association studies have shown that a number of common variants increase the risk of developing OA. Investigating their activity can uncover novel causal pathways and potentially highlight new treatment targets. One of the reported OA association signals is marked by the single nucleotide polymorphism (SNP) rs11842874 at chromosome 13q34. rs11842874 is positioned within a small linkage disequilibrium (LD) block within intron 4 of MCF2L, a gene encoding guanine-nucleotide exchange factor DBS. There are no non-synonymous SNPs that correlate with this association signal and we therefore set out to assess whether its effect on OA susceptibility is mediated by alteration of MCF2L expression. METHODS: Nucleic acid was extracted from cartilage, synovial membrane or infrapatellar fat pad tissues from OA patients. Expression of MCF2L was measured by quantitative PCR and RNA-sequencing whilst the presence of DBS was studied using immunohistochemistry. The functional effect of SNPs within the 13q34 locus was assessed using public databases and in vitro using luciferase reporter analysis. RESULTS: MCF2L gene and protein expression are detectable in joint tissues, with quantitative differences in the expression of the gene and in the transcript isoforms expressed between the tissues tested. There is an expression quantitative trait locus (eQTL) operating within synovial membrane tissue, with possession of the risk-conferring A allele of rs11842874 correlating with increased MCF2L expression. SNPs within the rs11842874 LD block reside within transcriptional regulatory elements and their direct analysis reveals that several show quantitative differences in regulatory activity at the allelic level. CONCLUSIONS: MCF2L is subject to a cis-acting eQTL in synovial membrane that correlates with the OA association signal. This signal contains several functional SNPs that could account for the susceptibility and which therefore merit further investigation. As far as we are aware, this is the first example of an OA susceptibility locus operating as an eQTL in synovial membrane tissue but not in cartilage.

Identification of gene regulatory networks affected across drug-resistant epilepsies.

Epilepsy is a chronic and heterogenous disease characterized by recurrent unprovoked seizures, that are commonly resistant to antiseizure medications. This study applies a transcriptome network-based approach across epilepsies aiming to improve understanding of molecular disease pathobiology, recognize affected biological mechanisms and apply causal reasoning to identify therapeutic hypotheses. This study included the most common drug-resistant epilepsies (DREs), such as temporal lobe epilepsy with hippocampal sclerosis (TLE-HS), and mTOR pathway-related malformations of cortical development (mTORopathies). This systematic comparison characterized the global molecular signature of epilepsies, elucidating the key underlying mechanisms of disease pathology including neurotransmission and synaptic plasticity, brain extracellular matrix and energy metabolism. In addition, specific dysregulations in neuroinflammation and oligodendrocyte function were observed in TLE-HS and mTORopathies, respectively. The aforementioned mechanisms are proposed as molecular hallmarks of DRE with the identified upstream regulators offering opportunities for drug-target discovery and development.

Mapping flows of embodied emissions in the global production system.

Environmentally extended multiregional input-output (MRIO) analysis can be used to investigate final production and consumption attributions of emissions. As the distinction between the two attributions has been brought to the attention of policy-makers, there is an ever greater need to understand how and why they differ, by analyzing the connections between production and consumption activities. Seeking to meet this need, we present an approach for mapping flows of embodied emissions through a Leontief production system. The approach, seen as an extension of Structural Path Analysis (SPA), provides an exhaustive map of supply chain linkages between final production and consumption attributions of emissions. Whereas SPA is traditionally used to extract and rank individual supply chains according to the emissions occurring at the end of each chain, the mapping approach considers emissions embodied in the flows of intermediate products linking different economic sectors along supply chains. Illustrative results are presented from a global MRIO model and CO(2) emissions for 2004. The emissions embodied in a sector's total output of products is also of interest: a method for calculating this is presented and shown to provide further insight into where in the production system a sector's overall emissions impact is concentrated.

DNA hypomethylation during MSC chondrogenesis occurs predominantly at enhancer regions.

Regulation of transcription occurs in a cell type specific manner orchestrated by epigenetic mechanisms including DNA methylation. Methylation changes may also play a key role in lineage specification during stem cell differentiation. To further our understanding of epigenetic regulation in chondrocytes we characterised the DNA methylation changes during chondrogenesis of mesenchymal stem cells (MSCs) by Infinium 450 K methylation array. Significant DNA hypomethylation was identified during chondrogenic differentiation including changes at many key cartilage gene loci. Integration with chondrogenesis gene expression data revealed an enrichment of significant CpGs in upregulated genes, while characterisation of significant CpG loci indicated their predominant localisation to enhancer regions. Comparison with methylation profiles of other tissues, including healthy and diseased adult cartilage, identified chondrocyte-specific regions of hypomethylation and the overlap with differentially methylated CpGs in osteoarthritis. Taken together we have associated DNA methylation levels with the chondrocyte phenotype. The consequences of which has potential to improve cartilage generation for tissue engineering purposes and also to provide context for observed methylation changes in cartilage diseases such as osteoarthritis.