Haploinsufficiency of A20 impairs protein–protein interactome and leads into caspase-8-dependent enhancement of NLRP3 inflammasome activation

Objectives TNFAIP3 encodes A20 that negatively regulates nuclear factor kappa light chain enhancer of activated B cells (NF-κB), the major transcription factor coordinating inflammatory gene expression. TNFAIP3 polymorphisms have been linked with a spectrum of inflammatory and autoimmune diseases and, recently, loss-of-function mutations in A20 were found to cause a novel inflammatory disease ‘haploinsufficiency of A20’ (HA20). Here we describe a family with HA20 caused by a novel TNFAIP3 loss-of-function mutation and elucidate the upstream molecular mechanisms linking HA20 to dysregulation of NF-κB and the related inflammasome pathway.Methods NF-κB activation was studied in a mutation-expressing cell line using luciferase reporter assay. Physical and close-proximity protein–protein interactions of wild-type and TNFAIP3 p.(Lys91*) mutant A20 were analysed using mass spectrometry. NF-κB -dependent transcription, cytokine secretion and inflammasome activation were compared in immune cells of the HA20 patients and control subjects.Results The protein–protein interactome of p.(Lys91*) mutant A20 was severely impaired, including interactions with proteins regulating NF-κB activation, DNA repair responses and the NLR family pyrin domain containing 3 (NLRP3) inflammasome. The p.(Lys91*) mutant A20 failed to suppress NF-κB signalling, which led to increased NF-κB -dependent proinflammatory cytokine transcription. Functional experiments in the HA20 patients’ immune cells uncovered a novel caspase-8-dependent mechanism of NLRP3 inflammasome hyperresponsiveness that mediated the excessive secretion of interleukin-1β and interleukin-18.Conclusions The current findings significantly deepen our understanding of the molecular mechanisms underlying HA20 and other diseases associated with reduced A20 expression or function, paving the way for future therapeutic targeting of the pathway.Peer reviewe

Validating 10-joint juvenile arthritis disease activity score cut-offs for disease activity levels in non-systemic juvenile idiopathic arthritis

Objectives To validate cut-offs of the Juvenile Arthritis Disease Activity Score 10 (JADAS10) and clinical JADAS10 (cJADAS10) and to compare them with other patient cohorts. Methods In a national multicentre study, cross-sectional data on recent visits of 337 non-systemic patients with juvenile idiopathic arthritis (JIA) were collected from nine paediatric outpatient units. The cut-offs were tested with receiver operating characteristic curve-based methods, and too high, too low and correct classification rates (CCRs) were calculated. Results Our earlier presented JADAS10 cut-offs seemed feasible based on the CCRs, but the cut-off values between low disease activity (LDA) and moderate disease activity (MDA) were adjusted. When JADAS10 cut-offs for clinically inactive disease (CID) were increased to 1.5 for patients with oligoarticular disease and 2.7 for patients with polyarticular disease, as recently suggested in a large multinational register study, altogether 11 patients classified as CID by the cut-off had one active joint. We suggest JADAS10 cut-off values for oligoarticular/polyarticular disease to be in CID: 0.0-0.5/0.0-0.7, LDA: 0.6-3.8/0.8-5.1 and MDA: >3.8/5.1. Suitable cJADAS10 cut-offs are the same as JADAS10 cut-offs in oligoarticular disease. In polyarticular disease, cJADAS10 cut-offs are 0-0.7 for CID, 0.8-5.0 for LDA and > 5.0 for MDA. Conclusion I nternational consensus on JADAS cutoff values is needed, and such a cut-off for CID should preferably exclude patients with active joints in the CID group.Peer reviewe

Pain-coping scale for children and their parents : a cross-sectional study in children with musculoskeletal pain

BackgroundIn a chronic pain-causing disease such as juvenile idiopathic arthritis, the quality of coping with pain is crucial. Parents have a substantial influence on their children's pain-coping strategies. This study aimed to develop scales for assessing parents' strategies for coping with their children's pain and a shorter improved scale for children usable in clinical practice.MethodsThe number of items in the Finnish version of the pain-coping questionnaire for children was reduced from 39 to 20. A corresponding reduced scale was created for parental use. We recruited consecutive patients from nine hospitals evenly distributed throughout Finland, aged 8-16 years who visited a paediatric rheumatology outpatient clinic and reported musculoskeletal pain during the past week. The patients and parents rated the child's pain on a visual analogue scale from 0 to 100 and completed pain-coping questionnaires and depression inventories. The selection process of pain questionnaire items was performed using factor analyses.ResultsThe average (standard deviation) age of the 130 patients was 13.0 (2.3) years; 91 (70%) were girls. Four factors were retained in the new, improved Pain-Coping Scales for children and parents. Both scales had 15 items with 2-5 items/factor. The goodness-of-fit statistics and Cronbach's alpha reliability coefficients were satisfactory to good in both scaled. The criterion validity was acceptable as the demographic, disease related, and the depression and stress questionnaires correlated with the subscales.ConclusionsWe created a shorter, feasible pain-coping scale for children and a novel scale for caregivers. In clinical work, the pain coping scales may serve as a visualisation of different types of coping strategies for paediatric patients with pain and their parents and facilitate the identification of families in need of psychological support.Peer reviewe

Heterozygous premature termination in zinc-finger domain of Kruppel-like factor 2 gene associates with dysregulated immunity

Kruppel-like factor 2 (KLF2) is a transcription factor with significant roles in development, maturation, differentiation, and proliferation of several cell types. In immune cells, KLF2 regulates maturation and trafficking of lymphocytes and monocytes. KLF2 participates in regulation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappa B) pathway. Although pulmonary arterial hypertension (PAH) related to KLF2 genetic variant has been suggested, genetic role of KLF2 associated with immune dysregulation has not been described. We identified a family whose members suffered from lymphopenia, autoimmunity, and malignancy. Whole exome sequencing revealed a KLF2 p.(Glu318Argfs*87) mutation disrupting the highly conserved zinc finger domain. We show a reduced amount of KLF2 protein, defective nuclear localization and altered protein-protein interactome. The phenotypically variable positive cases presented with B and T cell lymphopenia and abnormalities in B and T cell maturation including low naive T cell counts and low CD27(+)IgD(-)IgM(-) switched memory B cells. KLF2 target gene (CD62L) expression was affected. Although the percentage of (CD25(+)FOXP3(+), CD25(+)CD127(-)) regulatory T cells (Treg) was high, the naive Treg cells (CD45RA(+)) were absent. Serum IgG1 levels were low and findings in one case were consistent with common variable immunodeficiency (CVID). Transcription of NF-kappa beta pathway genes and p65/RelA phosphorylation were not significantly affected. Inflammasome activity, transcription of genes related with JAK/STAT pathway and interferon signature were also comparable to controls. Evidence of PAH was not found. In conclusion, KLF2 variant may be associated with familial immune dysregulation. Although the KLF2 deficient family members in our study suffered from lymphopenia, autoimmunity or malignancy, additional study cohorts are required to confirm our observations.Peer reviewe

Defining new clinically derived criteria for high disease activity in non-systemic juvenile idiopathic arthritis: a Finnish multicentre study

ObjectivesTo redefine criteria for high disease activity (HDA) in JIA, to establish HDA cut-off values for the 10-joint Juvenile Arthritis Disease Activity Score (JADAS10) and clinical JADAS10 (cJADAS10) and to describe the distribution of patients’ disease activity levels based on the JADAS cut-off values in the literature.MethodsData on 305 treatment-naïve JIA patients were collected from nine paediatric units treating JIA. The median parameters of the JADAS were proposed to be the clinical criteria for HDA. The cut-off values were assessed by using two receiver operating characteristics curve–based methods. The patients were divided into disease activity levels based on currently used JADAS cut-off values.ResultsWe proposed new criteria for HDA. At least three of the following criteria must be satisfied in both disease courses: in oligoarthritis, two or more active joints, ESR above normal, physician global assessment (PGA) of disease activity ≥2 and parent/patient global assessment (PtGA) of well-being ≥2; in polyarthritis, six or more active joints, ESR above normal, PGA of overall disease activity ≥4 and PtGA of well-being ≥2. The HDA cut-off values for JADAS10 (cJADAS) were ≥6.7 (6.7) for oligoarticular and ≥15.3 (14.1) for polyarticular disease. The distribution of the disease activity levels based on the JADAS cut-off values in the literature varied markedly based on which cut-offs were used.ConclusionNew clinically derived criteria for HDA in JIA and both JADAS and cJADAS cut-off values for HDA were proposed.</div

Validating 10-joint juvenile arthritis disease activity score cut-offs for disease activity levels in non-systemic juvenile idiopathic arthritis

Objectives To validate cut-offs of the Juvenile Arthritis Disease Activity Score 10 (JADAS10) and clinical JADAS10 (cJADAS10) and to compare them with other patient cohorts.Methods In a national multicentre study, cross-sectional data on recent visits of 337 non-systemic patients with juvenile idiopathic arthritis (JIA) were collected from nine paediatric outpatient units. The cut-offs were tested with receiver operating characteristic curve-based methods, and too high, too low and correct classification rates (CCRs) were calculated.Results Our earlier presented JADAS10 cut-offs seemed feasible based on the CCRs, but the cut-off values between low disease activity (LDA) and moderate disease activity (MDA) were adjusted. When JADAS10 cut-offs for clinically inactive disease (CID) were increased to 1.5 for patients with oligoarticular disease and 2.7 for patients with polyarticular disease, as recently suggested in a large multinational register study, altogether 11 patients classified as CID by the cut-off had one active joint. We suggest JADAS10 cut-off values for oligoarticular/polyarticular disease to be in CID: 0.0–0.5/0.0–0.7, LDA: 0.6–3.8/0.8–5.1 and MDA: >3.8/5.1. Suitable cJADAS10 cut-offs are the same as JADAS10 cut-offs in oligoarticular disease. In polyarticular disease, cJADAS10 cut-offs are 0–0.7 for CID, 0.8–5.0 for LDA and >5.0 for MDA.Conclusion International consensus on JADAS cut-off values is needed, and such a cut-off for CID should preferably exclude patients with active joints in the CID group.</div

Proceedings of the 24th Paediatric Rheumatology European Society Congress: Part three

From Springer Nature via Jisc Publications Router.Publication status: PublishedHistory: collection 2017-09, epub 2017-09-0

Myeloid-related protein 8/14 in plasma and serum in patients with new-onset juvenile idiopathic arthritis in real-world setting in a single center

Abstract Objective: The aim of this study was to analyze the usefulness of myeloid-related protein 8/14 (MRP8/14) in the prediction of disease course in a real-world setting for patients with new-onset juvenile idiopathic arthritis (JIA), to identify the relationship between MRP8/14 and disease activity using the physician’s global assessment of disease activity (PGA), and determine whether the MRP8/14 levels measured in serum and plasma are equally useful. Methods: In this prospective follow-up study, 87 new-onset non-systemic JIA patients were studied. Blood and synovial fluid samples were collected prior to any antirheumatic medication use. MRP8/14 was measured from serum (S-MRP8/14), plasma (P-MRP8/14), and synovial fluid samples using ELISA. Results: The baseline MRP8/14 blood levels were significantly higher in patients using synthetic antirheumatic drugs than in patients with no systemic medications at 1 year after diagnosis in serum (mean 298 vs. 198 ng/ml, P &lt; 0.001) and in plasma (mean 291 vs. 137 ng/ml, P = 0.001). MRP8/14 levels at the time of JIA diagnosis were higher in patients who started methotrexate during 1.5-year follow-up compared to those who achieved long-lasting inactive disease status without systemic medications (serum: mean 298 vs. 219 ng/ml, P = 0.006 and plasma: 296 vs. 141 ng/ml, P = 0.001). P-MRP8/14 was the most effective predictive variable for disease activity (by PGA) in linear multivariate regression model (combined to ESR, CRP, leukocytes, and neutrophils), whereas S-MRP8/14 was not significant. Conclusion: Blood MRP8/14 levels at baseline seem to predict disease course in new-onset JIA patients. P-MRP8/14 might be better than S-MRP8/14 when assessing disease activity at the time of JIA diagnosis