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Single nucleotide polymorphisms (SNP) near theERAP2gene are associated with autoimmune conditions such asCrohn’s disease, andbirdshot chorioretinopathy, as well as protection against lethal infections, including theBlack Death. Due to high linkage disequilibrium (LD), a great number of trait-associated SNPs are correlated withERAP2expression, however their functional mechanisms remain unidentified. We used genome editing and functional genomics to identify causal variants that remain obscured by LD. We demonstrate by reciprocal allelic replacement thatERAP2expression is directly controlled by the genotype of splice region SNP rs2248374. However, we demonstrate that autoimmune disease-risk SNPs located near the downstreamLNPEPgene promoter are independently associated withERAP2expression. Allele-specific conformation capture assays revealed long-range chromatin contacts between theLNPEPpromoter region and theERAP2promoter and showed that interactions were stronger in patients carrying the alleles that increase susceptibility to autoimmune diseases. Replacing the disease-associated SNPs in theLNPEPpromoter by reference sequences loweredERAP2expression. These findings show that clustered GWAS signals associated with diverse autoimmune conditions and lethal infections act in concert to control ERAP2 expression and that disease-associated variants can convert a gene promoter region into a potent enhancer of a distal gene.

Dermal fibroblasts are strategically positioned underneath the basal epidermis layer to support keratinocyte proliferation and extracellular matrix production. In inflammatory conditions, these fibroblasts produce cytokines and chemokines that promote the chemoattraction of immune cells into the dermis and the hyperplasia of the epidermis, two characteristic hallmarks of psoriasis. However, how dermal fibroblasts specifically contribute to psoriasis development remains largely uncharacterized. In this study, we investigated through which cytokines and signaling pathways dermal fibroblasts contribute to the inflammatory features of psoriatic skin. We show that dermal fibroblasts from lesional psoriatic skin are important producers of inflammatory mediators, including IL-6, CXCL8, and CXCL2. This increased cytokine production was found to be regulated by ZFP36 family members ZFP36, ZFP36L1, and ZFP36L2, RNA-binding proteins with mRNA-degrading properties. In addition, the expression of ZFP36 family proteins was found to be reduced in chronic inflammatory conditions that mimic psoriatic lesional skin. Collectively, these results indicate that dermal fibroblasts are important producers of cytokines in psoriatic skin and that reduced expression of ZFP36 members in psoriasis dermal fibroblasts contributes to their inflammatory phenotype.

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.

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.

Background Involving the end-users of scientific research (patients, carers and clinicians) in setting research priorities is important to formulate research questions that truly make a difference and are in tune with the needs of patients. We therefore aimed to generate a national research agenda for Juvenile Idiopathic Arthritis (JIA) together with patients, their caregivers and healthcare professionals through conducting a nationwide survey among these stakeholders. Methods The James Lind Alliance method was used, tailored with additional focus groups held to involve younger patients. First, research questions were gathered through an online and hardcopy survey. The received questions that were in scope were summarised and a literature search was performed to verify that questions were unanswered. Questions were ranked in the interim survey, and the final top 10 was chosen during a prioritisation workshop. Results Two hundred and seventy-eight respondents submitted 604 questions, of which 519 were in scope. Of these 604 questions, 81 were generated in the focus groups with younger children. The questions were summarised into 53 summary questions. An evidence checking process verified that all questions were unanswered. A total of 303 respondents prioritised the questions in the interim survey. Focus groups with children generated a top 5 of their most important questions. Combining this top 5 with the top 10s of patients, carers, and clinicians led to a top 21. Out of these, the top 10 research priorities were chosen during a final workshop. Research into pain and fatigue, personalised treatment strategies and aetiology were ranked high in the Top 10. Conclusions Through this study, the top 10 research priorities for JIA of patients, their caregivers and clinicians were identified to inform researchers and research funders of the research topics that matter most to them. The top priority involves the treatment and mechanisms behind persisting pain and fatigue when the disease is in remission.

ObjectiveTregs are crucial for immune regulation, and environment-driven adaptation of effector (e)Tregs is essential for local functioning. However, the extent of human Treg heterogeneity in inflammatory settings is unclear.MethodsWe combined single-cell RNA- and TCR-sequencing on Tregs derived from 4-6 patients with juvenile idiopathic arthritis (JIA) to investigate the functional heterogeneity of human synovial fluid (SF)-derived Tregs from inflamed joints. Confirmation and suppressive function of the identified Treg clusters was assessed by flow cytometry.ResultsFour Treg clusters were identified; incoming, activated eTregs with either a dominant suppressive or cytotoxic profile, and GPR56+CD161+CXCL13+ Tregs. Pseudotime analysis showed differentiation towards either classical eTreg profiles or GPR56+CD161+CXCL13+ Tregs supported by TCR data. Despite its most differentiated phenotype GPR56+CD161+CXCL13+ Tregs were shown to be suppressive. Furthermore, BATF was identified as an overarching eTreg regulator, with the novel Treg-associated regulon BHLHE40 driving differentiation towards GPR56+CD161+CXCL13+ Tregs, and JAZF1 towards classical eTregs.ConclusionOur study reveals a heterogeneous population of Tregs at the site of inflammation in JIA. SF Treg differentiate to a classical eTreg profile with a more dominant suppressive or cytotoxic profile that share a similar TCR repertoire, or towards GPR56+CD161+CXCL13+ Tregs with a more distinct TCR repertoire. Genes characterizing GPR56+CD161+CXCL13+ Tregs were also mirrored in other T-cell subsets in both the tumor and autoimmune setting. Finally, the identified key regulators driving SF Treg adaptation may be interesting targets for autoimmunity or tumor interventions.

Upon antigen-specific T Cell Receptor (TCR) engagement, human CD4+ T cells proliferate and differentiate, a process associated with rapid transcriptional changes and metabolic reprogramming. Here, we show that generation of extra-mitochondrial pyruvate is an essential step for acetyl-CoA production and subsequent H3K27ac-mediated epigenome remodeling. In contrast, neither acetate/ACSS2 nor citrate/ACLY metabolism are required for activation-induced transcriptional changes. Furthermore, T cell activation results in the nuclear translocation of PDC and its association with both the p300 acetyltransferase and histone H3K27ac. These data support tight integration of metabolic and histone-modifying enzymes, allowing metabolic reprogramming to fuel CD4+ T cell activation. Targeting this pathway may provide a novel therapeutic approach to specifically regulate antigen-driven T cell activation.

Dendritic cells (DC) are crucial for initiating and shaping immune responses. So far, little is known about heterogeneity and functional specialization of human DC subsets in (local) inflammatory conditions. We profiled conventional (c)DC1, cDC2 and monocytes based on phenotype, transcriptome and function from a local inflammatory site, namely synovial fluid (SF) from patients suffering from a chronic inflammatory condition, Juvenile Idiopathic Arthritis (JIA). cDC1, a relatively small dendritic cell subset in blood, are strongly enriched in SF. SF cDC1 showed a quiescent immune signature without a clear inflammatory profile low expression of PRR, chemokine, and cytokine receptors, and poor induction of T cell proliferation and cytokine production, but selective production of IFNλ upon polyinosinic:polycytidylic acid exposure. In stark contrast, cDC2 and monocytes from the same environment, showed a pro-inflammatory transcriptional profile, high levels of (spontaneous) pro-inflammatory cytokine production, and strong induction of T cell proliferation and cytokine production, including IL-17. Although the cDC2 and monocytes showed an overlapping transcriptional core profile, there were clear differences in the transcriptional landscape and functional features, indicating that these cell types retain their lineage identity in chronic inflammatory conditions. In conclusion, our findings suggest that at the site of inflammation, there is specific functional programming of human DC subsets, especially cDC2. In contrast, the enriched subset of cDC1 remains relatively quiescent and seemingly unchanged under inflammatory conditions, pointing to a more regulatory role.

T-cell resilience is critical to the immune pathogenesis of human autoimmune arthritis. Autophagy is essential for memory T cell generation and associated with pathogenesis in rheumatoid arthritis (RA). Our aim here was to delineate the role and molecular mechanism of autophagy in resilience and persistence of pathogenic T cells from autoimmune arthritis. We demonstrated "Autophagic memory" as elevated autophagy levels in CD4+ memory T cells compared to CD4+ naive T cells and in Jurkat Human T cell line trained with starvation stress. We then showed increased levels of autophagy in pathogenic CD4+ T cells subsets from autoimmune arthritis patients. Using RNA-sequencing, transcription factor gene regulatory network and methylation analyses we identified MYC as a key regulator of autophagic memory. We validated MYC levels using qPCR and further demonstrated that inhibiting MYC increased autophagy. The present study proposes the novel concept of autophagic memory and suggests that autophagic memory confers metabolic advantage to pathogenic T cells from arthritis and supports its resilience and long term survival. Particularly, suppression of MYC imparted the heightened autophagy levels in pathogenic T cells. These studies have a direct translational valency as they identify autophagy and its metabolic controllers as a novel therapeutic target.

Treg are critical regulators of immune homeostasis, and increasing evidence demonstrates that environment-driven Treg differentiation into effector (e)Treg is crucial for optimal functioning. However, human Treg programming under inflammatory conditions remains poorly understood. Here, we combine transcriptional and epigenetic profiling to identify the human eTreg core signature. Functional autoimmune inflammation-derived Treg display a unique transcriptional profile characterized by upregulation of both a core Treg (FOXP3, CTLA-4, TIGIT) and effector program (GITR, BLIMP-1, BATF). We identified a specific human eTreg signature that includes the vitamin D receptor (VDR) as predicted key-regulator in eTreg differentiation. H3K27ac/H3K4me1 occupancy revealed pronounced changes in the (super-)enhancer landscape, including enrichment of the binding motif for VDR and BATF. The observed Treg profile showed striking overlap with tumor-infiltrating Treg. Our data demonstrate that human inflammation-derived Treg acquire a specific eTreg profile guided by epigenetic changes. The core eTreg profile is conserved, and fine-tuned by environment-specific adaptations.

When a T cell and an antigen-presenting cell form an immunological synapse, rapid dynein-driven translocation of the centrosome towards the contact site leads to reorganization of microtubules and associated organelles. Currently, little is known about how the regulation of microtubule dynamics contributes to this process. Here, we show that the knockout of KIF21B, a kinesin-4 linked to autoimmune disorders, causes microtubule overgrowth and perturbs centrosome translocation. KIF21B restricts microtubule length by inducing microtubule pausing typically followed by catastrophe. Catastrophe induction with vinblastine prevented microtubule overgrowth and was sufficient to rescue centrosome polarization in KIF21B-knockout cells. Biophysical simulations showed that a relatively small number of KIF21B molecules can restrict microtubule length and promote an imbalance of dynein-mediated pulling forces that allows the centrosome to translocate past the nucleus. We conclude that proper control of microtubule length is important for allowing rapid remodeling of the cytoskeleton and efficient T cell polarization.

Objective: Systemic juvenile idiopathic arthritis (JIA) is a multifactorial autoinflammatory disease with a historically poor prognosis. With current treatment regimens, approximately half of patients still experience active disease after 1 year of therapy. This study was undertaken to evaluate a treat-to-target approach using recombinant interleukin-1 receptor antagonist (rIL-1Ra; anakinra) as first-line monotherapy to achieve early inactive disease and prevent damage. Methods: In this single-center, prospective study, patients with new-onset systemic JIA with an unsatisfactory response to nonsteroidal antiinflammatory drugs received rIL-1Ra monotherapy according to a treat-to-target strategy. Patients with an incomplete response to 2 mg/kg rIL-1Ra subsequently received 4 mg/kg rIL-1Ra or additional prednisolone, or switched to alternative therapy. For patients in whom inactive disease was achieved, rIL-1Ra was tapered after 3 months and subsequently stopped. Results: Forty-two patients, including 12 who had no arthritis at disease onset, were followed up for a median of 5.8 years. The median time to achieve inactive disease was 33 days. At 1 year, 76% had inactive disease, and 52% had inactive disease while not receiving medication. High neutrophil counts at baseline and a complete response after 1 month of rIL-1Ra were highly associated with inactive disease at 1 year. After 5 years of follow-up, 96% of the patients included had inactive disease, and 75% had inactive disease while not receiving medication. Articular or extraarticular damage was reported in

Juvenile Idiopathic Arthritis (JIA) is characterized by a loss of immune tolerance. Here, the balance between the activity of effector T (Teff) cells and regulatory T (Treg) cells is disturbed resulting in chronic inflammation in the joints. Presently, therapeutic strategies are predominantly aimed at suppressing immune activation and pro-inflammatory effector mechanisms, ignoring the opportunity to also promote tolerance by boosting the regulatory side of the immune balance. Histone deacetylases (HDACs) can deacetylate both histone and non-histone proteins and have been demonstrated to modulate epigenetic regulation as well as cellular signaling in various cell types. Importantly, HDACs are potent regulators of both Teff cell and Treg cell function and can thus be regarded as attractive therapeutic targets in chronic inflammatory arthritis. HDAC inhibitors (HDACi) have proven therapeutic potential in the cancer field, and are presently being explored for their potential in the treatment of autoimmune diseases. Specific HDACi have already been demonstrated to reduce the secretion of pro-inflammatory cytokines by Teff cells, and promote Treg numbers and suppressive capacity in vitro and in vivo. In this review, we outline the role of the different classes of HDACs in both Teff cell and Treg cell function. Furthermore, we will review the effect of different HDACi on T cell tolerance and explore their potential as a therapeutic strategy for the treatment of oligoarticular and polyarticular JIA.

Rheumatoid arthritis (RA) is an autoimmune disease hallmarked by aberrant cellular homeostasis, resulting in hyperactive CD4+ T cells that are more resistant to apoptosis. Both hyperactivation and resistance to apoptosis may contribute to the pathogenicity of CD4+ T cells in the autoimmune process. A better knowledge of the mechanisms determining such impaired homeostasis could contribute significantly to both the understanding and the treatment of the disease. Here we investigated whether autophagy, is dysregulated in CD4+ T cells of RA patients, resulting in disturbed T-cell homeostasis. We demonstrate that the rate of autophagy is significantly increased in CD4+ T cells from RA patients, and that increased autophagy is also a feature of in vitro activated CD4+ T cells. The increased apoptosis resistance observed in CD4+ T cells from RA patients was significantly reversed upon autophagy inhibition. These mechanisms may contribute to RA pathogenesis, as autophagy inhibition reduced both arthritis incidence and disease severity in a mouse collagen induced arthritis mouse model. Conversely, in Atg5flox/flox -CD4-Cre+ mice, in which all T cells are autophagy deficient, T cells showed impaired activation and proliferation. These data provide novel insight into the pathogenesis of RA and underscore the relevance of autophagy as a promising therapeutic target.

T cell factor, the effector transcription factor of the WNT signaling pathway, was so named because of the primary observation that it is indispensable for T cell development in the thymus. Since this discovery, the role of this signaling pathway has been extensively studied in T cell development, hematopoiesis, and stem cells; however, its functional role in mature T cells has remained relatively underinvestigated. Over the last few years, various studies have demonstrated that T cell factor can directly influence T cell function and the differentiation of Th1, Th2, Th17, regulatory T cell, follicular helper CD4+ T cell subsets, and CD8+ memory T cells. In this paper, we discuss the molecular mechanisms underlying these observations and place them in the general context of immune responses. Furthermore, we explore the implications and limitations of these findings for WNT manipulation as a therapeutic approach for treating immune-related diseases.

Contains fulltext : 200061.pdf (Publisher’s version ) (Open Access) BACKGROUND: Research on Juvenile Idiopathic Arthritis (JIA) should support patients, caregivers/parents (carers) and clinicians to make important decisions in the consulting room and eventually to improve the lives of patients with JIA. Thus far these end-users of JIA-research have rarely been involved in the prioritisation of future research. MAIN BODY: Dutch organisations of patients, carers and clinicians will collaboratively develop a research agenda for JIA, following the James Lind Alliance (JLA) methodology. In a 'Priority Setting Partnership' (PSP), they will gradually establish a top 10 list of the most important unanswered research questions for JIA. In this process the input from clinicians, patients and their carers will be equally valued. Additionally, focus groups will be organised to involve young people with JIA. The involvement of all contributors will be monitored and evaluated. In this manner, the project will contribute to the growing body of literature on how to involve young people in agenda setting in a meaningful way. CONCLUSION: A JIA research agenda established through the JLA method and thus co-created by patients, carers and clinicians will inform researchers and research funders about the most important research questions for JIA. This will lead to research that really matters.

Background One of the key elements of immune pathogenesis of human autoimmune arthritis is the resilience of pathogenic T cells. We have previously described that CD4 +T cells in patients with arthritis have an increased level of autophagy than their healthy equivalents. Here, we sought to explore at epigenetic and transcriptional levels the concept of persisting increased autophagy as the consequence of “autophagic memory”, as one of the mechanisms conferring resilience to pathogenic T cells, in particular to a subset of CD4 +T cells (CPL: Circulating Pathogenic-like Lymphocytes), which are significantly more represented in patients with active arthritis and resistant to therapy with biologics Objectives To understand molecular mechanism of resilience and persistence in pathogenic T cells in rheumatoid arthritis. Methods Autophagy in T cells were analysed using CytoID autophagy detection kit. Jurkat cells pre-starved and control were harvested at various time points, and RNA was extracted for RNA-sequencing and DNA methylation analysis. Illumina paired end sequencing was performed and data was analysed using open source tools in R statistical programming software. CD4 +memory and naive T cells were sorted using flow cytometer for qPCR analysis. The CD4 +memory and naive cells were sorted using Flow cytometer. RNA extracted and converted to cDNA for qPCR analysis of key genes Results First, we demonstrated elevated autophagic levels in CD4 +memory T cells when compared to naive CD4 +T cells. Second, we showed that autophagic levels are increased in naive and CD4 +T cells from RA patients compared to healthy controls. Using next generation RNA-sequencing, transcription factor gene regulatory network (TF-GRN) and methylation analyses, we identified MYC as key regulator of autophagic memory in a human T cell line. Transcriptome and network analysis of RNA-seq data from patients’ CPLs confirmed MYC as key modulator of autophagy. Importantly, inhibitor of MYC increases autophagy Conclusions The present study suggests that autophagic memory is retained both at the transcriptional and epigenetic levels as an integral part of mechanisms of efficient activation and survival of memory T cells. This mechanism is particularly relevant for cells subsets, such as CPLs, which are relevant to the immunopathogenesis of autoimmune diseases, such as arthritis. These studies have a direct translational valency as they identify autophagy and its metabolic controllers as a novel therapeutic target. Disclosure of Interest None declared

In the past decades, we have gained important insights into the mechanisms of disease and therapy underlying chronic inflammation in juvenile idiopathic arthritis (JIA). These insights have resulted in several game-changing therapeutic modalities for many patients. However, additional progress still has to be made with regard to efficacy, cost reduction, minimization of side effects, and dose-tapering and stop strategies of maintenance drugs. Moreover, to really transform the current therapeutic strategies into personalized medicine, we need validated biomarkers to translate increased insights into clinical practice. In this article, we describe recent developments in JIA research and outline how clinical innovations need to go hand in hand with basic discoveries to really effect care for patients. Facilitating the transition from bench to bedside is crucial for addressing the major current challenges in JIA management. When successful, it will set new standards for a safe, targeted, and personalized medicine in JIA.

C/EBPε, a member of the CCAAT/enhancer binding protein (C/EBP) family of transcription factors, is exclusively expressed in myeloid cells and regulates transition from the promyelocytic stage to the myelocytic stage of neutrophil development, being indispensable for secondary and tertiary granule formation. Knowledge concerning the functional role of C/EBPε posttranslational modifications is limited to studies concerning phosphorylation and sumoylation. In the current study, using ectopic expression and ex vivo differentiation of CD34(+) hematopoietic progenitor cells, we demonstrate that C/EBPε is acetylated, which was confirmed by mass spectrometry analysis, identifying 4 acetylated lysines in 3 distinct functional domains. Regulation of C/EBPε acetylation levels by the p300 acetyltransferase and the sirtuin 1 deacetylase controls transcriptional activity, which can at least in part be explained by modulation of DNA binding. During neutrophil development, acetylation of lysines 121 and 198 were found to be crucial for terminal neutrophil differentiation and the expression of neutrophil-specific granule proteins, including lactoferrin and collagenase. Taken together, our data illustrate a critical role for acetylation in the functional regulation of C/EBPε activity during terminal neutrophil development.

Objectives Systemic immunological processes are profoundly shaped by the micro-environments where antigen recognition occurs. Identifying molecular signatures distinctive of such processes is pivotal to understand pathogenic immune responses and manipulate them for therapeutic purposes. Unfortunately, direct investigation of peripheral tissues, enriched in pathogenic T cells, is often impossible or imposingly invasive in humans. Conversely, blood is easily accessible, but pathogenic signatures are diluted systemically as a result of the strict compartmentalisation of immune responses. In this work, we aimed at defining immune mediators shared between the bloodstream and the synovial micro-environment, and relevant for disease activity in autoimmune arthritis. Methods CD4 + T cells from blood and synovium of patients with juvenile idiopathic arthritis (JIA) were immunophenotyped by flow cytometry. The TCR repertoire of a circulating subset showing similarity with the synovium was analysed through next-generation sequencing of TCR β-chain CDR3 to confirm enrichment in synovial clonotypes. Finally, clinical relevance was established by monitoring the size of this subset in the blood of patients with JIA and rheumatoid arthritis (RA). Results We identified a small subset of circulating CD4 + T cells replicating the phenotypical signature of lymphocytes infiltrating the inflamed synovium. These circulating pathogenic-like lymphocytes (CPLs) were enriched in synovial clonotypes and they exhibited strong production of pro-inflammatory cytokines. Importantly, CPLs were expanded in patients with JIA, who did not respond to therapy, and also correlated with disease activity in patients with RA. Conclusions CPLs provide an accessible reservoir of pathogenic cells recirculating into the bloodstream and correlating with disease activity, to be exploited for diagnostic and research purposes.

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.

Forkhead box (FOX)P3 is a requisite transcription factor for the development and maintenance of immunosuppressive function of regulatory T (Treg) cells, and therefore for immune homeostasis. Post-translational modifications (PTMs) can transiently alter the functionality of transcription factors, and recent evidence reveals that FOXP3 can be regulated by various PTMs including acetylation, ubiquitination, and phosphorylation. Here, we review the current understanding of how these modifications control FOXP3, including regulation of DNA binding, transactivation potential, and proteasomal degradation. We place these findings in the context of the biology of Treg cells, and discuss both limitations in translating biochemical findings into in vivo functions and the opportunities presented by a better understanding of the molecular mechanisms that can transiently control FOXP3 activity in response to environmental cues.

Objectives JIA is an autoimmune disease involving disturbed T-cell homeostasis, marked by highly activated effector T cells. Autophagy, a lysosomal degradation pathway, is crucial for maintaining cellular homeostasis by regulating the survival, differentiation and function of a large variety of cells, including T cells. The aim of this study was to examine the rate of autophagy in JIA T cells and to investigate the effect of inhibition of autophagy on the inflammatory phenotype of JIA T cells. Methods Autophagy-related gene expression was analysed in CD4+ T cells from the SF of JIA patients and healthy controls using RNA sequencing. Autophagy was measured by flow cytometry and western blot. The effect of inhibition of autophagy, using HCQ, on the cellular activation status was analysed using flow cytometry and multiplex immunoassay. Results Autophagy was increased in T cells derived from the site of inflammation compared with cells from the peripheral blood of patients and healthy controls. This increase in autophagy was not induced by JIA SF, but is more likely to be the result of increased cellular activation. Inhibition of autophagy reduced proliferation, cytokine production and activation marker expression of JIA SF-derived CD4+ T cells. Conclusion These data indicate that autophagy is increased in JIA SF-derived T cells and that targeting autophagy could be a promising therapeutic strategy to restore the disrupted T-cell homeostasis in JIA.

For many autoimmune diseases, the underlying mechanism is still unknown. In order to get more insight into the etiology of autoimmune diseases, we recently published a study were we performed epigenetic profiling and RNA sequencing on CD4+CD45RO+ T cells derived from the site of inflammation of Juvenile Idiopathic Arthritis (JIA) patients and compared this with healthy controls [1]. In this “Data in Brief”, we focus on the analysis of our RNA sequencing data reported in this study, of which the raw and processed files can be found in GEO under GSE71595. We provide a detailed description of the downstream analysis, quality controls, and different analysis methods or techniques that validate the results obtained with RNA-sequencing. Keywords: Autoimmune disease, RNA-sequencing, Juvenile Idiopathic Arthritis, BET-inhibition

During the last decade research has focused on the application of FOXP3+ regulatory T cells (Tregs) in the treatment of autoimmune disease. However, thorough functional characterization of these cells in patients with chronic autoimmune disease, especially at the site of inflammation, is still missing. Here we studied Treg function in patients with juvenile idiopathic arthritis (JIA) and observed that Tregs from the peripheral blood as well as the inflamed joints are fully functional. Nevertheless, Treg-mediated suppression of cell proliferation and cytokine production by effector cells from the site of inflammation was severely impaired, because of resistance to suppression. This resistance to suppression was not caused by a memory phenotype of effector T cells or activation status of antigen presenting cells. Instead, activation of protein kinase B (PKB)/c-akt was enhanced in inflammatory effector cells, at least partially in response to TNFα and IL-6, and inhibition of this kinase restored responsiveness to suppression. We are the first to show that PKB/c-akt hyperactivation causes resistance of effector cells to suppression in human autoimmune disease. Furthermore, these findings suggest that for a Treg enhancing strategy to be successful in the treatment of autoimmune inflammation, resistance because of PKB/c-akt hyperactivation should be targeted as well.

Regulatory T cells (Tregs) are a specific subset of lymphocytes that are critical for the maintenance of self-tolerance. Expression levels of the transcription factor Foxp3 have been causally associated with Treg differentiation and function. Recent studies show that Foxp3 can also be transiently expressed in effector T cells; however, stable Foxp3 expression is required for development of a functional Treg suppressor phenotype. Here, we demonstrate that Foxp3 is acetylated, and this can be reciprocally regulated by the histone acetyltransferase p300 and the histone deacetylase SIRT1. Hyperacetylation of Foxp3 prevented polyubiquitination and proteasomal degradation, therefore dramatically increasing stable Foxp3 protein levels. Moreover, using mouse splenocytes, human peripheral blood mononuclear cells, T cell clones, and skin-derived T cells, we demonstrate that treatment with histone deacetylase inhibitors resulted in significantly increased numbers of functional Treg cells. Taken together, our data demonstrate that modulation of the acetylation state of Foxp3 provides a novel molecular mechanism for assuring rapid temporal control of Foxp3 levels in T cells, thereby regulating Treg numbers and functionality. Manipulating Foxp3 acetylation levels could therefore provide a new therapeutic strategy to control inappropriate (auto)immune responses.

Transcriptional coregulators, including the acetyltransferase Tip60, have a key role in complex cellular processes such as differentiation. Whereas post-translational modifications have emerged as an important mechanism to regulate transcriptional coregulator activity, the identification of the corresponding demodifying enzymes has remained elusive. Here we show that the expression of the Tip60 protein, which is essential for adipocyte differentiation, is regulated through polyubiquitination on multiple residues. USP7, a dominant deubiquitinating enzyme in 3T3-L1 adipocytes and mouse adipose tissue, deubiquitinates Tip60 both in intact cells and in vitro and increases Tip60 protein levels. Furthermore, inhibition of USP7 expression and activity decreases adipogenesis. Transcriptome analysis reveals several cell cycle genes to be co-regulated by both Tip60 and USP7. Knockdown of either factor results in impaired mitotic clonal expansion, an early step in adipogenesis. These results reveal deubiquitination of a transcriptional coregulator to be a key mechanism in the regulation of early adipogenesis.

SummaryStable Foxp3 expression is required for the development of functional regulatory T (Treg) cells. Here, we demonstrate that the expression of the transcription factor Foxp3 can be regulated through the polyubiquitination of multiple lysine residues, resulting in proteasome-mediated degradation. Expression of the deubiquitinase (DUB) USP7 was found to be upregulated and active in Treg cells, being associated with Foxp3 in the nucleus. Ectopic expression of USP7 decreased Foxp3 polyubiquitination and increased Foxp3 expression. Conversely, either treatment with DUB inhibitor or USP7 knockdown decreased endogenous Foxp3 protein expression and decreased Treg-cell-mediated suppression in vitro. Furthermore, in a murine adoptive-transfer-induced colitis model, either inhibition of DUB activity or USP7 knockdown in Treg cells abrogated their ability to resolve inflammation in vivo. Our data reveal a molecular mechanism in which rapid temporal control of Foxp3 expression in Treg cells can be regulated by USP7, thereby modulating Treg cell numbers and function.

Maintenance of Foxp3 protein expression in regulatory T cells (Treg) is crucial for a balanced immune response. We have previously demonstrated that Foxp3 protein stability can be regulated through acetylation, however the specific mechanisms underlying this observation remain unclear. Here we demonstrate that SIRT1 a member of the lysine deacetylase Sirtuin (SIRT) family, but not the related SIRTs 2–7, co-localize with Foxp3 in the nucleus. Ectopic expression of SIRT1, but not SIRTs 2–7 results in decreased Foxp3 acetylation, while conversely inhibition of endogenous SIRT activity increased Foxp3 acetylation. We show that SIRT1 inhibition decreases Foxp3 poly-ubiquitination, thereby increasing Foxp3 protein levels. Co-transfection of SIRT1 with Foxp3 results in increased Foxp3 proteasomal degradation, while SIRT inhibition increases FOXP3 transcriptional activity in human Treg. Taken together, these data support a central role for SIRT1 in the regulation of Foxp3 protein levels and thereby in regulation of Treg suppressive capacity. Pharmacological modulation of SIRT1 activity in Treg may therefore provide a novel therapeutic strategy for controlling immune responses.