Stable 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.
Display omitted
•DUBs regulate Foxp3 protein expression through the inhibition of degradation•Expression of the DUB USP7 is selectively upregulated during Treg cell differentiation•Foxp3 protein expression is increased by USP7-mediated Foxp3 deubiquitination•USP7 modulates Treg-cell-mediated suppression both in vitro and in vivo
Foxp3 is crucial for both the development and function of regulatory T (Treg) cells; however, the posttranslational mechanisms regulating Foxp3 transcriptional output remain poorly defined. Here, we ...demonstrate that T cell factor 1 (TCF1) and Foxp3 associates in Treg cells and that active Wnt signaling disrupts Foxp3 transcriptional activity. A global chromatin immunoprecipitation sequencing comparison in Treg cells revealed considerable overlap between Foxp3 and Wnt target genes. The activation of Wnt signaling reduced Treg-mediated suppression both in vitro and in vivo, whereas disruption of Wnt signaling in Treg cells enhanced their suppressive capacity. The activation of effector T cells increased Wnt3a production, and Wnt3a levels were found to be greatly increased in mononuclear cells isolated from synovial fluid versus peripheral blood of arthritis patients. We propose a model in which Wnt produced under inflammatory conditions represses Treg cell function, allowing a productive immune response, but, if uncontrolled, could lead to the development of autoimmunity.
•TCF/β-catenin and Foxp3 share common transcriptional targets•Wnt signaling negatively modulates Foxp3 transcriptional activity•Wnt signaling reduces Treg-cell-mediated suppression in vitro and in vivo•Wnt production is increased upon the activation of T cells and at inflammatory loci
Growing knowledge about cellular interactions in the immune system, including the central role of cytokine networks, has lead to new treatments using monoclonal antibodies that block specific ...components of the immune system. Systemic cytokine concentrations can serve as surrogate outcome parameters of these interventions to study inflammatory pathways operative in patients in vivo. This is now possible due to novel technologies such as multiplex immunoassays (MIA) that allows detection of multiple cytokines in a single sample. However, apparently trivial underappreciated processes, (sample handling and storage, interference of endogenous plasma proteins) can greatly impact the reliability and reproducibility of cytokine detection.Therefore we set out to investigate several processes that might impact cytokine profiles such as blood collecting tubes, duration of storage, and number of freeze thawing cycles.
Since under physiological conditions cytokine concentrations normally are low or undetectable we spiked cytokines in the various plasma and serum samples. Overall recoveries ranged between 80-120%. Long time storage showed cytokines are stable for a period up to 2 years of storage at -80 degrees C. After 4 years several cytokines (IL-1alpha, IL-1beta, IL-10, IL-15 and CXCL8) degraded up to 75% or less of baseline values. Furthermore we show that only 2 out of 15 cytokines remained stable after several freeze-thawing cycles. We also demonstrate implementation of an internal control for multiplex cytokine immunoassays.
All together we show parameters which are essential for measurement of cytokines in the context of clinical trials.
Macrophages contribute significantly to the pathology of many chronic inflammatory diseases, including rheumatoid arthritis (RA), asthma, and chronic obstructive pulmonary disease. Macrophage ...activation and survival are tightly regulated by reversible acetylation and deacetylation of histones, transcription factors, and structural proteins. Although histone deacetylase (HDAC) inhibitors (HDACis) demonstrate therapeutic effects in animal models of chronic inflammatory disease, depressed macrophage HDAC activity in patients with asthma, chronic obstructive pulmonary disease, or RA may contribute to inflammation in these diseases, potentially contraindicating the therapeutic administration of HDACis. In this study, we directly examined whether HDACis could influence the activation of macrophages derived from the inflamed joints of patients with RA. We found that inhibition of class I/II HDACs or class III sirtuin HDACs potently blocked the production of IL-6 and TNF-alpha by macrophages from healthy donors and patients with RA. Two HDACis, trichostatin A and nicotinamide, selectively induced macrophage apoptosis associated with specific downregulation of the antiapoptotic protein Bfl-1/A1, and inflammatory stimuli enhanced the sensitivity of macrophages to HDACi-induced apoptosis. Importantly, inflammatory and angiogenic cytokine production in intact RA synovial biopsy explants was also suppressed by HDACis. Our study identifies redundant, but essential, roles for class I/II and sirtuin HDACs in promoting inflammation, angiogenesis, and cell survival in RA.
Objective
To conduct a prospective cohort study using anakinra, a recombinant IL‐1 receptor antagonist (IL‐1Ra), as first‐line therapy in patients with new‐onset systemic juvenile idiopathic ...arthritis (JIA).
Methods
Therapy with recombinant IL‐1Ra (2 mg/kg) was initiated in 20 patients who fulfilled the International League of Associations for Rheumatology criteria for systemic JIA, before systemic steroid treatment was administered. Patients were monitored clinically and immunologically. The protocol contained a stop strategy for patients who met at least the adapted American College of Rheumatology 90% criteria for improvement in JIA (ACR Pediatric 90 ACR Pedi 90) after 3 months.
Results
We included consecutive patients with new‐onset systemic JIA. The mean followup period was 32 months (range 12–54 months). At the 3‐month time point, 85% of the patients showed an adapted ACR Pedi 90 response or had inactive disease; 75% of the patients achieved this response while receiving recombinant IL‐1Ra alone. After 1 year, 17 of the 20 patients met the criteria for clinically inactive disease, and 13 of these patients met these criteria while receiving monotherapy with recombinant IL‐1Ra. However, because of persistent disease activity, 7 of the 20 patients required additional therapy besides recombinant IL‐1Ra. According to our stop strategy, 73% of patients with at least an adapted ACR Pedi 90 response at 3 months could stop recombinant IL‐1Ra treatment within 1 year. After 2 years, 12 (86%) of 14 patients met the criteria for disease remission, either while receiving (n = 4) or not receiving (n = 8) medication. After 3 years, 10 (91%) of 11 patients met the criteria for disease remission, either while receiving (n = 2) or not receiving (n = 8) medication.
Conclusion
This is the first prospective study in which recombinant IL‐1Ra was used as first‐line therapy in patients with systemic JIA. We observed excellent responses in nearly all patients within 3 months. In the majority of responding patients, treatment with recombinant IL‐1Ra could be stopped within 1 year, with remission being preserved during followup. In approximately one‐third of patients, concomitant therapy was required for maintenance of clinical response.
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.
The underlying molecular mechanisms for many autoimmune diseases are poorly understood. Juvenile idiopathic arthritis (JIA) is an exceptionally well-suited model for studying autoimmune diseases due ...to its early onset and the possibility to analyze cells derived from the site of inflammation. Epigenetic profiling, utilizing primary JIA patient-derived cells, can contribute to the understanding of autoimmune diseases. With H3K27ac chromatin immunoprecipitation, we identified a disease-specific, inflammation-associated, typical enhancer and super-enhancer signature in JIA patient synovial-fluid-derived CD4+ memory/effector T cells. RNA sequencing of autoinflammatory site-derived patient T cells revealed that BET inhibition, utilizing JQ1, inhibited immune-related super-enhancers and preferentially reduced disease-associated gene expression, including cytokine-related processes. Altogether, these results demonstrate the potential use of enhancer profiling to identify disease mediators and provide evidence for BET inhibition as a possible therapeutic approach for the treatment of autoimmune diseases.
Display omitted
•Identification of a disease-specific, inflammation-associated, enhancer signature•Inflammatory-site-derived cells are epigenetically different from peripheral cells•Enrichment of disease-related SNPs in disease-associated super-enhancers•BET inhibition of JIA patient T cells preferentially reduced JIA gene expression
By defining the active super-enhancer profile of autoimmune disease patients, Peeters et al. identify a disease-specific, inflammation-associated super-enhancer signature. In addition, inhibition of super-enhancer activity, using a BET inhibitor, in autoimmune disease patient-derived cells preferentially reduced disease-associated gene expression. These findings suggest a role for enhancers and super-enhancers in autoimmune diseases and demonstrates the potential use of BET inhibitors for the treatment of such diseases.
Chronic inflammatory diseases are characterized by recurrent inflammatory attacks in the tissues mediated by autoreactive T cells. Identity and functional programming of CD8+ T cells at the target ...site of inflammation still remain elusive. One key question is whether, in these antigen-rich environments, chronic stimulation leads to CD8+ T cell exhaustion comparable to what is observed in infectious disease contexts. In the synovial fluid (SF) of juvenile idiopathic arthritis (JIA) patients, a model of chronic inflammation, an overrepresentation of PD-1+CD8+ T cells was found. Gene expression profiling, gene set enrichment analysis, functional studies, and extracellular flux analysis identified PD-1+CD8+ T cells as metabolically active effectors, with no sign of exhaustion. Furthermore, PD-1+CD8+ T cells were enriched for a tissue-resident memory (Trm) cell transcriptional profile and demonstrated increased clonal expansion compared with the PD-1- counterpart, suggesting antigen-driven expansion of locally adapted cells. Interestingly, this subset was also found increased in target tissues in other human chronic inflammatory diseases. These data indicate that local chronic inflammation drives the induction and expansion of CD8+ T cells endowed with potential detrimental properties. Together, these findings lay the basis for investigation of PD-1-expressing CD8+ T cell targeting strategies in human chronic inflammatory diseases.
Since the discovery of FOXP3+ regulatory T (T(REG)) cells over 15 years ago, intensive research has focused on their presence, phenotype and function in autoimmune disease. Whether deficiencies in ...T(REG) cells underlie autoimmune pathology and whether, or how, therapeutic approaches based on these cells might be successful is still the subject of debate. The potential role of T(REG)-cell extrinsic factors, such as proinflammatory cytokines and resistance of effector T cells to suppression, as the cause of regulatory defects in chronic autoimmune inflammation is an intensive area of research. It is now clear that, at the site of inflammation, antigen presenting cells (APCs) and proinflammatory cytokines drive effector T cell skewing and plasticity, and that these T cells can become unresponsive to regulation. In addition, expansion and function of T(REG) cells is affected by the inflammatory environment; indeed, new data suggest that, in certain conditions, T(REG) cells promote inflammation. This Review summarizes the latest findings on changes in effector T cell homeostasis in autoimmune disease and focuses on how mechanisms that normally regulate these cells are affected in the inflamed joints of patients with arthritis. These findings have important clinical implications and will affect the development of new therapeutic strategies for autoimmune arthritis.
PDF
