Treg cells are critical regulators of immune homeostasis, and environment-driven Treg cell differentiation into effector (e)Treg cells is crucial for optimal functioning. However, human Treg cell ...programming in inflammation is unclear. Here, we combine transcriptional and epigenetic profiling to identify a human eTreg cell signature. Inflammation-derived functional Treg cells have a transcriptional profile characterized by upregulation of both a core Treg cell (FOXP3, CTLA4, TIGIT) and effector program (GITR, BLIMP-1, BATF). We identify a specific human eTreg cell signature that includes the vitamin D receptor (VDR) as a predicted regulator in eTreg cell differentiation. H3K27ac/H3K4me1 occupancy indicates an altered (super-)enhancer landscape, including enrichment of the VDR and BATF binding motifs. The Treg cell profile has striking overlap with tumor-infiltrating Treg cells. Our data demonstrate that human inflammation-derived Treg cells acquire a conserved and specific eTreg cell profile guided by epigenetic changes, and fine-tuned by environment-specific adaptations.
Abstract
Objectives
How the local inflammatory environment regulates epigenetic changes in the context of inflammatory arthritis remains unclear. Here we assessed the transcriptional and active ...enhancer profile of monocytes derived from the inflamed joints of JIA patients, a model well-suited for studying inflammatory arthritis.
Methods
RNA sequencing and H3K27me3 chromatin immunoprecipitation sequencing (ChIP-seq) were used to analyse the transcriptional and epigenetic profile, respectively, of JIA synovial fluid-derived monocytes.
Results
Synovial-derived monocytes display an activated phenotype, which is regulated on the epigenetic level. IFN signalling-associated genes are increased and epigenetically altered in synovial monocytes, indicating a driving role for IFN in establishing the local inflammatory phenotype. Treatment of synovial monocytes with the Janus-associated kinase (JAK) inhibitor ruxolitinib, which inhibits IFN signalling, transformed the activated enhancer landscape and reduced disease-associated gene expression, thereby inhibiting the inflammatory phenotype.
Conclusion
This study provides novel insights into epigenetic regulation of inflammatory arthritis patient-derived monocytes and highlights the therapeutic potential of epigenetic modulation for the treatment of inflammatory rheumatic diseases.
Juvenile dermatomyositis (JDM) is an immune-mediated inflammatory disease affecting the microvasculature of skin and muscle. CD4+ CD25+ FOXP3+ regulatory T cells (Tregs) are key regulators of immune ...homeostasis. A role for Tregs in JDM pathogenesis has not yet been established. Here, we explored Treg presence and function in peripheral blood and muscle of JDM patients. We analyzed number, phenotype and function of Tregs in blood from JDM patients by flow cytometry and in vitro suppression assays, in comparison to healthy controls and disease controls (Duchenne's Muscular Dystrophy). Presence of Tregs in muscle was analyzed by immunohistochemistry. Overall, Treg percentages in peripheral blood of JDM patients were similar compared to both control groups. Muscle biopsies of new onset JDM patients showed increased infiltration of numbers of T cells compared to Duchenne's muscular dystrophy. Both in JDM and Duchenne's muscular dystrophy the proportion of FOXP3+ T cells in muscles were increased compared to JDM peripheral blood. Interestingly, JDM is not a self-remitting disease, suggesting that the high proportion of Tregs in inflamed muscle do not suppress inflammation. In line with this, peripheral blood Tregs of active JDM patients were less capable of suppressing effector T cell activation in vitro, compared to Tregs of JDM in clinical remission. These data show a functional impairment of Tregs in a proportion of patients with active disease, and suggest a regulatory role for Tregs in JDM inflammation.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Objective
Neutrophils are the most abundant innate immune cells in the blood, but little is known about their role in (acquired) chronic autoinflammatory diseases. This study was undertaken to ...investigate the role of neutrophils in systemic‐onset juvenile idiopathic arthritis (JIA), a prototypical multifactorial autoinflammatory disease that is characterized by arthritis and severe systemic inflammation.
Methods
Fifty patients with systemic‐onset JIA who were receiving treatment with recombinant interleukin‐1 receptor antagonist (rIL‐1Ra; anakinra) were analyzed at disease onset and during remission. RNA sequencing was performed on fluorescence‐activated cell–sorted neutrophils from 3 patients with active systemic‐onset JIA and 3 healthy controls. Expression of activation markers, apoptosis, production of reactive oxygen species (ROS), and degranulation of secretory vesicles from neutrophils were assessed by flow cytometry in serum samples from 17 patients with systemic‐onset JIA and 15 healthy controls.
Results
Neutrophil counts were markedly increased at disease onset, and this correlated with the levels of inflammatory mediators. The neutrophil counts normalized within days after the initiation of rIL‐1Ra therapy. RNA‐sequencing analysis revealed a substantial up‐regulation of inflammatory processes in neutrophils from patients with active systemic‐onset JIA, significantly overlapping with the transcriptome of sepsis. Correspondingly, neutrophils from patients with active systemic‐onset JIA displayed a primed phenotype that was characterized by increased ROS production, CD62L shedding, and secretory vesicle degranulation, which was reversed by rIL‐1Ra treatment in patients who had achieved clinical remission. Patients with a short disease duration had high neutrophil counts, more immature neutrophils, and a complete response to rIL‐1Ra, whereas patients with symptoms for >1 month had normal neutrophil counts and an unsatisfactory response to rIL‐1Ra. In vitro, rIL‐1Ra antagonized the priming effect of IL‐1β on neutrophils from healthy subjects.
Conclusion
These results strongly support the notion that neutrophils play an important role in systemic‐onset JIA, especially in the early inflammatory phase of the disease. The findings also demonstrate that neutrophil numbers and the inflammatory activity of systemic‐onset JIA are both susceptible to IL‐1 blockade.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Objective
Human leukocyte antigen (HLA)‐DRB1*15:01 has been recently associated with interstitial lung disease (LD), eosinophilia, and drug reactions in systemic juvenile idiopathic arthritis (sJIA). ...Additionally, genetic variants in IL1RN have been linked to poor response to anakinra. We sought to reproduce these findings in a prospective cohort study of patients with new‐onset sJIA treated with anakinra as first‐line therapy.
Methods
HLA and IL1RN risk alleles were identified via whole‐genome sequencing. Treatment responses and complications were compared between carriers versus noncarriers.
Results
Seventeen of 65 patients (26%) carried HLA‐DRB1*15:01, comparable with the general population, and there was enrichment for HLA‐DRB1*11:01, a known risk locus for sJIA. The rates of clinical inactive disease (CID) at 6 months, 1 year, and 2 years were generally high, irrespective of HLA‐DRB1 or IL1RN variants, but significantly lower in carriers of an HLA‐DRB1*11:01 allele. One patient, an HLA‐DRB1*15:01 carrier, developed sJIA‐LD. Of the three patients with severe drug reactions to biologics, one carried HLA‐DRB1*15:01. The prevalence of eosinophilia did not significantly differ between HLA‐DRB1*15:01 carriers and noncarriers at disease onset (6.2% vs 14.9%, P = 0.67) nor after the start of anakinra (35.3% vs 37.5% in the first 2 years of disease).
Conclusion
We observed high rates of CID using anakinra as first‐line treatment irrespective of HLA‐DRB1 or IL1RN variants. Only one of the 17 HLA‐DRB1*15:01 carriers developed sJIA‐LD, and of the three patients with drug reactions to biologics, only one carried HLA‐DRB1*15:01. Although thorough monitoring for the development of drug hypersensitivity and refractory disease courses in sJIA, including sJIA‐LD, remains important, our data support the early start of biologic therapy in patients with new‐onset sJIA irrespective of HLA‐DRB1 background or IL1RN variants.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
N6-methyladenosine (m6A) is a RNA modification that can regulate post-transcriptional processes including RNA stability, translation, splicing and nuclear export. In CD4+ lymphocytes, m6A ...modifications have been demonstrated to play a role in early differentiation processes. The role of m6A in CD4+ T cell activation and effector function remains incompletely understood. To assess the role of m6A in CD4+ T lymphocyte activation and function, we assessed the transcriptome-wide m6A landscape of human primary CD4+ T cells by methylated RNA immunoprecipitation (meRIP) sequencing. Stimulation of the T cells impacted the m6A pattern of hundreds of transcripts including tumor necrosis factor (TNF). m6A methylation was increased on TNF mRNA after activation, predominantly in the 3' untranslated region (UTR) of the transcript. Manipulation of m6A levels in primary human T cells, the directly affected the expression of TNF. Furthermore, we identified that the m6A reader protein YT521-B homology domain family-2 (YTHDF2) binds m6A-methylated TNF mRNA, and promotes its degradation. Taken together, this study demonstrates that TNF expression in CD4+ T lymphocytes is regulated via m6A and YTHDF2, thereby providing novel insight into the regulation of T cell effector functions.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Respiratory syncytial virus (RSV) is the most prevalent cause of acute lower respiratory infection in young children. Currently, the first RSV vaccines are approved by the FDA. Recently, ...N6-methyladenosine (m6A) RNA methylation has been implicated in the regulation of the viral life cycle and replication of many viruses, including RSV. m6A methylation of RSV RNA has been demonstrated to promote replication and prevent anti-viral immune responses by the host. Whether m6A is also involved in viral entry and whether m6A can also affect RSV infection via different mechanisms than methylation of viral RNA is poorly understood. Here, we identify m6A reader YTH domain-containing protein 1 (YTHDC1) as a novel negative regulator of RSV infection. We demonstrate that YTHDC1 abrogates RSV infection by reducing the expression of RSV entry receptor CX3C motif chemokine receptor 1 (CX3CR1) on the cell surface of lung epithelial cells. Altogether, these data reveal a novel role for m6A methylation and YTHDC1 in the viral entry of RSV. These findings may contribute to the development of novel treatment options to control RSV infection.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
•In house validation shows inter-assay variation <10%, and average inter-assay variation of 12.2%•Immune profiles remain stable after multiple freeze–thaw cycles.•Only 19 out of 162 soluble proteins ...have similar expression in serum, EDTA plasma and sodium heparin plasma.
Diagnosis of complex disease and response to treatment is often associated with multiple indicators, both clinical and laboratorial. With the use of biomarkers, various mechanisms have been unraveled which can lead to better and faster diagnosis, predicting and monitoring of response to treatment and new drug development. With the introduction of multiplex technology for immunoassays and the growing awareness of the role of immune-monitoring during new therapeutic interventions it is now possible to test large numbers of soluble mediators in small sample volumes. However, standardization of sample collection and laboratory assessments remains suboptimal.
We developed a multiplex immunoassay for detection of 162 immune related proteins in human serum and plasma. The assay was split in panels depending on natural occurring concentrations with a maximum of 60 proteins. The aim of this study was to evaluate precision, accuracy, reproducibility and stability of proteins when repeated freeze–thaw cycles are performed of this in-house developed panel, as well as assessing the protein signature in plasma and serum using various anticoagulants.
Intra-assay variance of each mediator was <10%. Inter-assay variance ranged between 1.6 and 37% with an average of 12.2%. Recoveries were similar for all mediators (mean 99.8 ± 2.6%) with a range between 89–107%. Next we measured all mediators in serum, EDTA plasma and sodium heparin plasma of 43 healthy control donors. Of these markers only 19 showed similar expression profiles in the 3 different matrixes. Only 5 mediators were effected by multiple freeze-thawing cycles. Principal component analysis revealed different coagulants cluster separately and that sodium heparin shows the most consistent profile.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Tregs are crucial for maintaining maternal immunotolerance against the semiallogeneic fetus. We investigated the elusive transcriptional profile and functional adaptation of human uterine Tregs ...(uTregs) during pregnancy. Uterine biopsies, from placental bed (materno-fetal interface) and incision site (control) and blood were obtained from women with uncomplicated pregnancies undergoing cesarean section. Tregs and CD4+ non-Tregs were isolated for transcriptomic profiling by Cel-Seq2. Results were validated on protein and single cell levels by flow cytometry. Placental bed uTregs showed elevated expression of Treg signature markers, including FOXP3, CTLA-4, and TIGIT. Their transcriptional profile was indicative of late-stage effector Treg differentiation and chronic activation, with increased expression of immune checkpoints GITR, TNFR2, OX-40, and 4-1BB; genes associated with suppressive capacity (HAVCR2, IL10, LAYN, and PDCD1); and transcription factors MAF, PRDM1, BATF, and VDR. uTregs mirrored non-Treg Th1 polarization and tissue residency. The particular transcriptional signature of placental bed uTregs overlapped strongly with that of tumor-infiltrating Tregs and was remarkably pronounced at the placental bed compared with uterine control site. In conclusion, human uTregs acquire a differentiated effector Treg profile similar to tumor-infiltrating Tregs, specifically at the materno-fetal interface. This introduces the concept of site-specific transcriptional adaptation of Tregs within 1 organ.
Objective
Resistance of Teff cells to Treg cell–mediated suppression contributes to the breakdown of peripheral tolerance in the inflamed joints of patients with juvenile idiopathic arthritis (JIA). ...However, unanswered questions are whether this resistant phenotype is self‐sustained and whether CD8+ and CD4+ Teff cells share the same mechanism of resistance to suppression. We undertook this study to investigate intrinsic resistance of CD8+ Teff cells to suppression and to determine how this can be targeted therapeutically.
Methods
CD8+ or CD4+ Teff cells were cultured with or without antigen‐presenting cells (APCs) in Treg cell–dependent and –independent suppression assays. Synovial fluid (SF)–derived Teff cells were crosscultured with peripheral blood (PB) Treg cells from JIA patients or healthy controls. Tumor necrosis factor (TNF) or interferon‐γ (IFNγ) blocking agents were used to restore Teff cell responsiveness to suppression.
Results
Suppression of cell proliferation and cytokine production in CD8+ Teff cells from the SF of JIA patients was severely impaired compared to that in CD8+ Teff cells from the PB of JIA patients, regardless of the presence of APCs and CD4+ Teff cells. Similar to CD4+ Teff cells, impaired suppression of CD8+ Teff cells was shown to be an intrinsic feature of this cell population. While TNF blockade restored both CD8+ and CD4+ Teff cell susceptibility to suppression, autocrine release of IFNγ selectively sustained CD8+ Teff cell resistance, which could be relieved by IFNγ blockade.
Conclusion
Unlike CD4+ Teff cells, resistance of CD8+ Teff cells to suppression at the site of autoimmune inflammation is maintained by autocrine release of IFNγ, and blockade of IFNγ restores CD8+ Teff cell responsiveness to suppression. These findings indicate a potential therapeutic value of blocking IFNγ to restore immune regulation in JIA.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
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