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Objectives Extracellular vesicles (EVs) are abundant in body fluids, contributing to intercellular signalling by transferring cargo that includes microRNAs (miRs)—themselves implicated in pathobiology. For the first time we evaluated the potential of EV miRs to contribute diagnostic information in early RA, predict methotrexate (MTX) efficacy or shed light on the drug's mechanism of action. Methods Seven hundred and ninety-eight miRs isolated from serum-derived EVs of 46 patients with untreated RA, 23 with untreated polymyalgia rheumatica (PMR; inflammatory disease control group) and 12 in whom significant inflammatory disease had been excluded (non-inflammatory controls; NICs) were profiled (NanoString); the same measurements were made for RA patients after 6 months' MTX treatment. Analyses took multiple testing into account. Results Twenty-eight EV miRs were robustly differentially expressed between early RA (but not PMR) patients and NICs after correction for age and sex, suggesting discriminatory value. Cross-validated partial least squares-discriminant analysis also indicated the predictive potential of a distinct baseline EV miR signature with respect to MTX-induced remission at 6 months. The change in expression of 13 miRs over the course of MTX treatment differed significantly between responders and non-responders, and four of those exhibiting increased relative abundance amongst responders have known roles in regulating the pathogenic potential of synovial fibroblasts, namely miR-212-3p, miR-338-5p, miR-410-3p and miR-537. Conclusion Our data highlight the potential of serum EV miRs as diagnostic and therapeutic biomarkers, highlighting a novel potential mechanism by which MTX may exert its therapeutic effect in early RA that warrants further investigation. [ABSTRACT FROM AUTHOR]

Modest effect sizes have limited the clinical applicability of genetic associations with rheumatic diseases. Genetic risk scores (GRSs) have emerged as a promising solution to translate genetics into useful tools. In this review, we provide an overview of the recent literature on GRSs in rheumatic diseases. We describe six categories for which GRSs are used: (a) disease (outcome) prediction, (b) genetic commonalities between diseases, (c) disease differentiation, (d) interplay between genetics and environmental factors, (e) heritability and transferability, and (f) detecting causal relationships between traits. In our review of the literature, we identified current lacunas and opportunities for future work. First, the shortage of non-European genetic data restricts the application of many GRSs to European populations. Next, many GRSs are tested in settings enriched for cases that limit the transferability to real life. If intended for clinical application, GRSs are ideally tested in the relevant setting. Finally, there is much to elucidate regarding the co-occurrence of clinical traits to identify shared causal paths and elucidate relationships between the diseases. GRSs are useful instruments for this. Overall, the ever-continuing research on GRSs gives a hopeful outlook into the future of GRSs and indicates significant progress in their potential applications. [ABSTRACT FROM AUTHOR]

Age‐associated B cells (ABCs) are an immune cell subset linked to autoimmunity, infection and ageing, and whose pathophysiological importance was recently highlighted using single cell synovial tissue profiling. To elucidate their pathophysiological relevance, peripheral blood (PB) ABCs from early rheumatoid arthritis (eRA) patients naïve to disease‐modifying anti‐rheumatic drugs (DMARDs) were compared with their synovial fluid (SF) counterparts, and to PB ABCs from psoriatic arthritis patients and healthy controls. PB and SF B‐cell subsets were phenotyped by multi‐parameter flow cytometry, sorted and subjected to gene expression profiling (NanoString nCounter® Immunology V2 Panel) and functional characterization (stimulated cytokine measurements by immunoassay). PB ABCs of eRA patients, which are transcriptionally distinct from those of control cohorts, express chemokine receptors and adhesion molecules, such as CXCR3, that favour homing to inflammatory sites over lymphoid tissue. These cells are an activated, class‐switched B‐cell subset expressing high levels of HLA‐DR, co‐stimulatory molecules and T‐bet. Their secretion profile includes IL‐12p70 and IL‐23 but low levels of IL‐10. High surface expression of FcRL family members, including FcRL3, furthermore suggests a role for these cells in autoimmunity. Finally, and unlike in the periphery where they are rare, ABCs are the predominant B‐cell subsets in SF. These observations indicate the predilection of ABCs for inflammatory tissue in RA, where their propensity for antigen presentation and pro‐inflammatory phenotype may support autoimmune pathology. Their potential as a therapeutic target therefore warrants further study. [ABSTRACT FROM AUTHOR]

Abstract Background Pancreatic beta-cells are the target of an autoimmune attack in type 1 diabetes mellitus (T1DM). This is mediated in part by cytokines, such as interleukin (IL)-1β and interferon (IFN)-γ. These cytokines modify the expression of hundreds of genes, leading to beta-cell dysfunction and death by apoptosis. Several of these cytokine-induced genes are potentially regulated by the IL-1β-activated transcription factor (TF) nuclear factor (NF)-κB, and previous studies by our group have shown that cytokine-induced NF-κB activation is pro-apoptotic in beta-cells. To identify NF-κB-regulated gene networks in beta-cells we presently used a discriminant analysis-based approach to predict NF-κB responding genes on the basis of putative regulatory elements. Results The performance of linear and quadratic discriminant analysis (LDA, QDA) in identifying NF-κB-responding genes was examined on a dataset of 240 positive and negative examples of NF-κB regulation, using stratified cross-validation with an internal leave-one-out cross-validation (LOOCV) loop for automated feature selection and noise reduction. LDA performed slightly better than QDA, achieving 61% sensitivity, 91% specificity and 87% positive predictive value, and allowing the identification of 231, 251 and 580 NF-κB putative target genes in insulin-producing INS-1E cells, primary rat beta-cells and human pancreatic islets, respectively. Predicted NF-κB targets had a significant enrichment in genes regulated by cytokines (IL-1β or IL-1β + IFN-γ) and double stranded RNA (dsRNA), as compared to genes not regulated by these NF-κB-dependent stimuli. We increased the confidence of the predictions by selecting only evolutionary stable genes, i.e. genes with homologs predicted as NF-κB targets in rat, mouse, human and chimpanzee. Conclusion The present in silico analysis allowed us to identify novel regulatory targets of NF-κB using a supervised classification method based on putative binding motifs. This provides new insights into the gene networks regulating cytokine-induced beta-cell dysfunction and death.

Objective: Cytokines contribute to pancreatic beta-cell death in type 1 diabetes. This effect is mediated by complex gene networks that remain to be characterized. We presently utilized array analysis to define the global expression pattern of genes, including spliced variants, modified by the cytokines interleukin (IL)-1beta + interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha + IFN-gamma in primary rat beta-cells.Research Design and Methods: Fluorescence-activated cell sorter-purified rat beta-cells were exposed to IL-1beta + IFN-gamma or TNF-alpha + IFN-gamma for 6 or 24 h, and global gene expression was analyzed by microarray. Key results were confirmed by RT-PCR, and small-interfering RNAs were used to investigate the mechanistic role of novel and relevant transcription factors identified by pathway analysis. RESULTS Nearly 16,000 transcripts were detected as present in beta-cells, with temporal differences in the number of genes modulated by IL-1beta + IFNgamma or TNF-alpha + IFN-gamma. These cytokine combinations induced differential expression of inflammatory response genes, which is related to differential induction of IFN regulatory factor-7. Both treatments decreased the expression of genes involved in the maintenance of beta-cell phenotype and growth/regeneration. Cytokines induced hypoxia-inducible factor-alpha, which in this context has a proapoptotic role. Cytokines also modified the expression of >20 genes involved in RNA splicing, and exon array analysis showed cytokine-induced changes in alternative splicing of >50% of the cytokine-modified genes.Conclusions: The present study doubles the number of known genes expressed in primary beta-cells, modified or not by cytokines, and indicates the biological role for several novel cytokine-modified pathways in beta-cells. It also shows that cytokines modify alternative splicing in beta-cells, opening a new avenue of research for the field. [ABSTRACT FROM AUTHOR]

Objective: Endogenous retroelements (EREs) stimulate type 1 interferon (IFN-I) production but have not been explored as potential interferonogenic triggers in rheumatoid arthritis (RA). We investigated ERE expression in early RA (eRA), a period in which IFN-I levels are increased., Methods: ERE expression (long terminal repeat [LTR] 5, long interspersed nuclear element 1 [LINE-1], and short interspersed nuclear element [SINE]) in disease-modifying treatment-naïve eRA whole-blood and bulk synovial tissue samples was examined by reverse transcription-polymerase chain reaction and NanoString alongside IFN-α activity. Circulating lymphocyte subsets, including B cell subsets, from patients with eRA and early psoriatic arthritis (ePsA) were flow cytometrically sorted and similarly examined. Existing established RA and osteoarthritis (OA) synovial single-cell sequencing data were reinterrogated to identify repeat elements, and associations were explored., Results: There was significant coexpression of all ERE classes and IFNA in eRA synovial tissue samples (n = 22, P < 0.0001) and significant positive associations between whole-blood LINE-1 expression (n = 56) and circulating IFN-α protein (P = 0.018) and anti-cyclic citrullinated peptide (anti-CCP) titers (P < 0.0001). ERE expression was highest in circulating eRA B cells, particularly naïve B cells compared with ePsA, with possible ERE regulation by SAM and HD Domain Containing Deoxynucleoside Triphosphate Triphosphohydrolase 1 transcription (SAMDH1) implicated and associations with IFNA again observed. Finally, in established RA synovium, LTRs, particularly human endogenous retroviral sequence K (HERVK), were most increased in RA compared with OA, in which, for all synovial subsets (monocytes, B cells, T cells, and fibroblasts), ERE expression associated with increased IFN-I signaling (P < 0.001)., Conclusion: Peripheral blood and synovial ERE expression is examined for the first time in eRA, highlighting both a potential causal relationship between ERE and IFN-I production and an intriguing association with anti-CCP autoantibodies. This suggests EREs may contribute to RA pathophysiology with implications for future novel therapeutic strategies., (© 2024 The Author(s). Arthritis & Rheumatology published by Wiley Periodicals LLC on behalf of American College of Rheumatology.)

Objectives: An interferon (IFN) gene signature (IGS) is present in approximately 50% of early, treatment naive rheumatoid arthritis (eRA) patients where it has been shown to negatively impact initial response to treatment. We wished to validate this effect and explore potential mechanisms of action., Methods: In a multicentre inception cohort of eRA patients (n=191), we examined the whole blood IGS ( MxA, IFI44L, OAS1, IFI6, ISG15 ) with reference to circulating IFN proteins, clinical outcomes and epigenetic influences on circulating CD19+ B and CD4+ T lymphocytes., Results: We reproduced our previous findings demonstrating a raised baseline IGS. We additionally showed, for the first time, that the IGS in eRA reflects circulating IFN-α protein. Paired longitudinal analysis demonstrated a significant reduction between baseline and 6-month IGS and IFN-α levels (p<0.0001 for both). Despite this fall, a raised baseline IGS predicted worse 6-month clinical outcomes such as increased disease activity score (DAS-28, p=0.025) and lower likelihood of a good EULAR clinical response (p=0.034), which was independent of other conventional predictors of disease activity and clinical response. Molecular analysis of CD4+ T cells and CD19+ B cells demonstrated differentially methylated CPG sites and dysregulated expression of disease relevant genes, including PARP9, STAT1, and EPSTI1 , associated with baseline IGS/IFNα levels. Differentially methylated CPG sites implicated altered transcription factor binding in B cells (GATA3, ETSI, NFATC2, EZH2) and T cells (p300, HIF1α)., Conclusions: Our data suggest that, in eRA, IFN-α can cause a sustained, epigenetically mediated, pathogenic increase in lymphocyte activation and proliferation, and that the IGS is, therefore, a robust prognostic biomarker. Its persistent harmful effects provide a rationale for the initial therapeutic targeting of IFN-α in selected patients with eRA., Competing Interests: Competing interests: JDI discloses research grants from Pfizer, Janssen and GSK; conference support from Eli Lilly and Gilead; speaker/consulting fees from AbbVie, BMS, Gilead, Roche and UCB. FAHC discloses speaker fees from AstraZeneca.The remaining authors have no competing interests., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY. Published by BMJ.)

Beta cell dysfunction and death in type 1 diabetes mellitus (T1DM) is caused by direct contact with activated macrophages and T lymphocytes and by exposure to soluble mediators secreted by these cells, such as cytokines and nitric oxide. Cytokine-induced apoptosis depends on the expression of pro- and anti-apoptotic genes that remain to be characterized. Using microarray analyses, we identified several transcription factor and "effector" gene networks regulated by interleukin-1beta and/or interferon-gamma in beta cells. This suggests that beta cell fate following exposure to cytokines is a complex and highly regulated process, depending on the duration and severity of perturbation of key gene networks. In order to draw correct conclusions from these massive amounts of data, we need to utilize novel bioinformatics and statistical tools. Thus, we are presently performing in silico analysis for the localization of binding sites for the transcription factor NF-kappaB (previously shown to be pivotal for beta cell apoptosis) in 15 temporally related gene clusters, identified by time-course microarray analysis. In silico analysis is based on a broad range of computational techniques used to detect motifs in a DNA sequence corresponding to the binding site of a transcription factor. These computer-based findings must be validated by use of positive and negative controls, and by "ChIP on chip" analysis. Moreover, new statistical approaches are required to decrease false positive findings. These novel approaches will constitute a "proof of principle" for the integrated use of bioinformatics and functional genomics in the characterization of relevant cytokine-regulated beta cell gene networks leading to beta cell apoptosis in T1DM.