Simona Ursu

Th17 plasticity in human autoimmune arthritis is driven by the inflammatory environment

In several murine models of autoimmune arthritis, Th17 cells are the dominant initiators of inflammation. In human arthritis the majority of IL-17–secreting cells within the joint express a cytokine phenotype intermediate between Th17 and Th1. Here we show that Th17/1 cells from the joints of children with inflammatory arthritis express high levels of both Th17 and Th1 lineage-specific transcription factors, RORC2 and T-bet. Modeling the generation of Th17/1 in vitro, we show that Th17 cells “convert” to Th17/1 under conditions that mimic the disease site, namely low TGFβ and high IL-12 levels, whereas Th1 cells cannot convert to Th17. Th17/1 cells from the inflamed joint share T-cell receptor (TCR) clonality with Th17 cells, suggesting a shared clonal origin between Th17 and Th17/1 cells in arthritis. Using CD161, a lectin-like receptor that is a marker of human Th17, we show synovial Th17 and Th17/1 cells, and unexpectedly, a large proportion of Th1 cells express CD161. We provide evidence to support a Th17 origin for Th1 cells expressing CD161. In vitro, Th17 cells that convert to a Th1 phenotype maintain CD161 expression. In the joint CD161+ Th1 cells share features with Th17 cells, with shared TCR clonality, expression of RORC2 and CCR6 and response to IL-23, although they are IL-17 negative. We propose that the Th17 phenotype may be unstable and that Th17 cells may convert to Th17/1 and Th1 cells in human arthritis. Therefore therapies targeting the induction of Th17 cells could also attenuate Th17/1 and Th1 effector populations within the inflamed joint.

CD161 defines the subset of FoxP3+ T cells capable of producing proinflammatory cytokines

Regulatory FoxP3+CD4+ T cells (Treg) are vital for maintaining the balance between tolerance, adequate immune response, and autoimmunity. Despite this immunoregulatory role, it has been shown that Treg may also produce proinflammatory cytokines. Here we present a distinct population of Treg, defined by CD161 expression, as the major source of FoxP3+ Treg-derived proinflammatory cytokines. CD161+ Treg can be followed throughout development, from thymus and cord blood to healthy child and adult samples. CD161+ Treg display anergy, are suppressive in cocultures with conventional T cells (Tconv), and possess a predominantly demethylated Treg-specific demethylated region of the FOXP3 locus. In addition to the production of interleukin (IL) 17A, interferon γ, and IL-2, CD161+FoxP3+ cells share markers with Tconv, including expression of the transcription factors retinoic acid-related orphan receptor Cv2 (RORCv2) and T-cell-specific T-box transcription factor (Tbet). Expression of CD161 and enrichment for cytokine production are stable characteristics of CD161+ Treg upon both short- and longer-term culture in vitro. Additionally, CD161+ Treg are highly enriched within the inflammatory environment of childhood arthritis, suggesting a role in disease. Our data therefore demonstrate that CD161+FoxP3+ T cells are a novel Treg subset, found in health and disease, which display high proinflammatory potential but also exhibit hallmark Treg characteristics.

The toll-like receptor 4 agonist MRP8/14 protein complex is a sensitive indicator for disease activity and predicts relapses in systemic-onset juvenile idiopathic arthritis

Background Analysis of myeloid-related protein 8 and 14 complex (MRP8/14) serum concentrations is a potential new tool to support the diagnosis of systemic-onset juvenile idiopathic arthritis (SJIA) in the presence of fever of unknown origin. Objective To test the ability of MRP8/14 serum concentrations to monitor disease activity in patients with SJIA and stratify patients at risk of relapse. Methods Serum concentrations of MRP8/14 in 52 patients with SJIA were determined by a sandwich ELISA. The monitoring of therapeutic regimens targeting interleukin 1 and tumour necrosis factor α, and methotrexate treatment was analysed and diagnostic power to predict flares was tested. Results MRP8/14 levels were clearly raised in active disease and decreased significantly in response to successful treatments. Serum concentrations of MRP8/14 increased significantly (p<0.001) (mean±95% CI 12.030±3.090 ng/ml) during disease flares compared with patients with inactive disease (864±86 ng/ml). During clinical remission MRP8/14 serum levels of >740 ng/ml predicted disease flares accurately (sensitivity 92%, specificity 88%). MRP8/14 levels correlated well with clinical disease activity, as assessed by physicians global assessment of disease activity (r=0.62), Childhood Health Assessment Questionnaire (r=0.56), active joint count (r=0.46) and with C-reactive protein (r=0.71) and erythrocyte sedimentation rate (r=0.72) (for all p<0.001). Conclusion MRP8/14 serum concentrations correlate closely with response to drug treatment and disease activity and therefore might be an additional measurement for monitoring anti-inflammatory treatment of individual patients with SJIA. MRP8/14 serum concentrations are the first predictive biomarker indicating subclinical disease activity and stratifying patients at risk of relapse during times of clinically inactive disease.

Association of the 5-aminoimidazole-4-carboxamide ribonucleotide transformylase gene with response to methotrexate in juvenile idiopathic arthritis

Objectives Methotrexate (MTX) is the mainstay treatment for juvenile idiopathic arthritis (JIA), however approximately 30% of children will fail to respond to the drug. Identification of genetic predictors of response to MTX would be invaluable in developing optimal treatment strategies for JIA. Using a candidate gene approach, single nucleotide polymorphisms (SNPs) within genes in the metabolic pathway of MTX, were investigated for association with response to treatment in JIA cases. Methods Tagging SNPs were selected across 13 MTX metabolic pathway genes and were genotyped using Sequenom genotyping technology in subjects recruited from the Sparks Childhood Arthritis Response to Medication Study. Response to MTX was defined using the American College of Rheumatology (ACR) paediatric response criteria and SNP genotype frequencies were compared between the worst and best responders (ACR-Ped70) to MTX. An independent cohort of US JIA cases was available for validation of initial findings. Results One SNP within the inosine triphosphate pyrophosphatase gene (ITPA) and two SNPs within 5-aminoimidazole-4-carboxamide ribonucleotide transformylase gene (ATIC) were significantly associated with a poor response to MTX. One of the ATIC SNPs showed a trend towards association with MTX response in an independent cohort of US JIA cases. Meta-analysis of the two studies strengthened this association (combined p value=0.002). Conclusions This study presents association of a SNP in the ATIC gene with response to MTX in JIA. There is now growing evidence to support a role of the ATIC gene with response to MTX treatment. These results could contribute towards a better understanding of and ability to predict MTX response in JIA.

A subgroup of juvenile idiopathic arthritis patients who respond well to methotrexate are identified by the serum biomarker MRP8/14 protein

OBJECTIVES In JIA there is an unmet need for biomarkers with which to identify patients who will respond well to MTX. The aim of this study was to define the prognostic value of baseline serum proteins and clinical variables in response to MTX to help inform the clinician at time of diagnosis whether the patient is likely to respond well to MTX. METHODS JIA patients were recruited into the Childhood Arthritis Response to Medication Study (CHARMS). Clinical data and venous blood were collected before administration of MTX and at follow-up. MRP8/14 and inflammatory cytokines were measured by ELISA and multiplex immunoassay, respectively. CRP and ESR were measured as part of routine clinical assessment. To explore which baseline factors might predict successful treatment, binary logistic regression models were fitted for outcome. RESULTS High disease activity (high serum MRP8/14, active joint count or physicians score) pre-MTX was observed in a subgroup of patients with a better response to therapy. In a multivariable analysis, after accounting for MRP8/14 at baseline, no other factors were independently significantly associated with outcome. Patients with baseline MRP8/14 >3000 ng/ml were more likely to respond to MTX at ACR50 or better: odds ratio 16.07 (95% CI 2.00, 129.3). CONCLUSION We have demonstrated that high levels of baseline serum MRP8/14 have prognostic value in predicting a subgroup of patients whose arthritis will improve on MTX. Routine collection of serum prior to the start of medication would be a valuable step in collaborative validation of such biomarkers.

Generation of novel pharmacogenomic candidates in response to methotrexate in juvenile idiopathic arthritis: correlation between gene expression and genotype.

Objectives Little is known about the mechanisms of efficacy of methotrexate (MTX) in childhood arthritis, or genetic influences upon response to MTX. The aims of this study were to use gene expression profiling to identify novel pathways/genes altered by MTX and then investigate these genes for genotype associations with response to MTX treatment. Methods Gene expression profiling before and after MTX treatment was performed on 11 children with juvenile idiopathic arthritis (JIA) treated with MTX, in whom response at 6 months of treatment was defined. Genes showing the most differential gene expression after the treatment were selected for single nucleotide polymorphism (SNP) genotyping. Genotype frequencies were compared between nonresponders and responders (ACR-Ped70). An independent cohort was available for validation. Results Gene expression profiling before and after MTX treatment revealed 1222 differentially expressed probes sets (fold change >1.7, P<0.05) and 1065 when restricted to full responder cases only. Six highly differentially expressed genes were analyzed for genetic association in response to MTX. Three SNPs in the SLC16A7 gene showed significant association with MTX response. One SNP showed validated association in an independent cohort. Conclusion This study is the first, to our knowledge, to evaluate gene expression profiles in children with JIA before and after MTX, and to analyze genetic variation in differentially expressed genes. We have identified a gene, which may contribute to genetic variability in MTX response in JIA, and established as proof of principle that genes that are differentially expressed at mRNA level after drug administration may also be good candidates for genetic analysis.

Th1 and Th17 cell subpopulations are enriched in the peripheral blood of patients with systemic juvenile idiopathic arthritis

Objective. The role of the adaptive immune system has not been explored in detail compared with the innate immune system in systemic JIA (sJIA) pathogenesis. The aim of this study was to examine the phenotype of circulating peripheral blood CD4+ T-cell subpopulations in a cross-sectional study of sJIA patients during disease remission on medication and during acute flare of the disease. Methods. Flow cytometry was used to examine the phenotype and cytokine production of IFNγ-, IL-4- and IL-17-producing CD4+ T cells in the peripheral blood of 10 sJIA patients with active disease, 9 sJIA with inactive disease, 14 JIA patients with oligoarticular onset, 10 adult control subjects and 10 age-matched control subjects. In parallel, we examined the proportion of FoxP3+ Tregs. Results. IFNγ- and IL-17-producing CD4+ T cells and IL-17-producing CD3+CD4− T cells were present at higher proportions in the peripheral blood of sJIA patients, irrespective of their disease status. Our data also confirm the known increase of the proportions of IFNγ-producing Th1 cells with increasing age and suggest an increase with age in the IL-17-producing CD4+ T-cell population. Conclusion. This study is the first to describe significantly higher proportions of Th1 and Th17 T helper cell subsets in the peripheral blood of sJIA patients. These proinflammatory cells may play a pathogenic role in sJIA. Our data also emphasize the importance of using paediatric age-matched control subjects when evaluating the T-cell cytokine profile in JIA.

Hypomethylation at the regulatory T cell-specific demethylated region in CD25hi T cells is decoupled from FOXP3 expression at the inflamed site in childhood arthritis.

The maintenance of FOXP3 expression in CD25hi regulatory T cells (Tregs) is crucial to the control of inflammation and essential for successful Treg transfer therapies. Coexpression of CD25 and FOXP3 in combination with a hypomethylated region within the FOXP3 gene, called the Treg-specific demethylated region (TSDR), is considered the hallmark of stable Tregs. The TSDR is an epigenetic motif that is important for stable FOXP3 expression and is used as a biomarker to measure Treg lineage commitment. In this study, we report that, unlike in peripheral blood, CD4+ T cell expression of CD25 and FOXP3 is frequently dissociated at the inflamed site in patients with juvenile idiopathic arthritis, which led us to question the stability of human Tregs in chronic inflammatory environments. We describe a novel CD4+CD127loCD25hi human T cell population that exhibits extensive TSDR and promoter demethylation in the absence of stable FOXP3 expression. This population expresses high levels of CTLA-4 and can suppress T conventional cell proliferation in vitro. These data collectively suggest that this population may represent a chronically activated FOXP3lo Treg population. We show that these cells have defects in IL-2 signaling and reduced expression of a deubiquitinase important for FOXP3 stability. Clinically, the proportions of these cells within the CD25hi T cell subset are increased in patients with the more severe courses of disease. Our study demonstrates, therefore, that hypomethylation at the TSDR can be decoupled from FOXP3 expression in human T cells and that environment-specific breakdown in FOXP3 stability may compromise the resolution of inflammation in juvenile idiopathic arthritis.

Genome-wide data reveal novel genes for methotrexate response in a large cohort of juvenile idiopathic arthritis cases.

Clinical response to methotrexate (MTX) treatment for children with juvenile idiopathic arthritis (JIA) displays considerable heterogeneity. Currently, there are no reliable predictors to identify non-responders: earlier identification could lead to a targeted treatment. We genotyped 759 JIA cases from the UK, the Netherlands and Czech Republic. Clinical variables were measured at baseline and 6 months after start of the treatment. In Phase I analysis, samples were analysed for the association with MTX response using ordinal regression of ACR-pedi categories and linear regression of change in clinical variables, and identified 31 genetic regions (P<0.001). Phase II analysis increased SNP density in the most strongly associated regions, identifying 14 regions (P<1 × 10−5): three contain genes of particular biological interest (ZMIZ1, TGIF1 and CFTR). These data suggest a role for novel pathways in MTX response and further investigations within associated regions will help to reach our goal of predicting response to MTX in JIA.

Association of the IL-10 Gene Family Locus on Chromosome 1 with Juvenile Idiopathic Arthritis (JIA)

Background The cytokine IL-10 and its family members have been implicated in autoimmune diseases and we have previously reported that genetic variants in IL-10 were associated with a rare group of diseases called juvenile idiopathic arthritis (JIA). The aim of this study was to fine map genetic variants within the IL-10 cytokine family cluster on chromosome 1 using linkage disequilibrium (LD)-tagging single nucleotide polymorphisms (tSNPs) approach with imputation and conditional analysis to test for disease associations. Methodology/Principal Findings Fifty-three tSNPs were tested for association between Caucasian paediatric cohorts [219 systemic JIA (sJIA), 187 persistent oligoarticular JIA (pOJIA), and 139 extended OJIA (eOJIA) patients], and controls (Wellcome Trust control cohort, WTCCC2). Significant association with sJIA was detected at rs1400986 in the promoter of IL-20 (odds ratio 1.53; 95% CI 1.21–1.93; p = 0.0004), but in no other subtypes. Imputation analysis identified additional associated SNPs for pOJIA at IL-20 and IL-24, including a rare, functional, missense variant at IL-24 with a p = 0.0002. Penalised logistic regression analysis with HyperLasso and conditional analysis identified several further associations with JIA subtypes. In particular, haplotype analysis refined the sJIA association, with a joint effect at rs1400986 and rs4129024 in intron 1 of MAPKAPK2 (p = 3.2E−5). For pOJIA, a 3-SNP haplotype including rs1878672 in intron 3 of IL-10 showed evidence for association (p = 0.0018). In eOJIA, rs10863962 (3′UTR of FCAMR) and rs12409577 (intron of IL-19) haplotype showed some evidence of association (p = 0.0003). Conclusions This study supports previous association of IL-20 with sJIA. Haplotype analyses provided stronger association signals than single point analyses, while a penalised logistic regression approach also suggested multiple independent association signals. Replication studies are required to confirm or refute these findings. The results indicate that combined effects with unknown/rare variants remain to be characterised in JIA, and represent a possible example of synthetic association in this region.