Halima Moncrieffe

Interleukin‐17–producing T cells are enriched in the joints of children with arthritis, but have a reciprocal relationship to regulatory T cell numbers

Objective To identify interleukin-17 (IL-17)–producing T cells from patients with juvenile idiopathic arthritis (JIA), and investigate their cytokine production, migratory capacity, and relationship to Treg cells at sites of inflammation, as well as to test the hypothesis that IL-17+ T cell numbers correlate with clinical phenotype in childhood arthritis. Methods Flow cytometry was used to analyze the phenotype, cytokine production, and chemokine receptor expression of IL-17–producing T cells in peripheral blood and synovial fluid mononuclear cells from 36 children with JIA, in parallel with analysis of forkhead box P3 (FoxP3)–positive Treg cells. Migration of IL-17+ T cells toward CCL20 was assessed by a Transwell assay. Synovial tissue was analyzed by immunohistochemistry for IL-17 and IL-22. Results IL-17+ T cells were enriched in the joints of children with JIA as compared with the blood of JIA patients (P = 0.0001) and controls (P = 0.018) and were demonstrated in synovial tissue. IL-17+ T cell numbers were higher in patients with extended oligoarthritis, the more severe subtype of JIA, as compared with patients with persistent oligoarthritis, the milder subtype (P = 0.046). Within the joint, there was an inverse relationship between IL-17+ T cells and FoxP3+ Treg cells (r = 0.61, P = 0.016). IL-17+,CD4+ T cells were uniformly CCR6+ and migrated toward CCL20, but synovial IL-17+ T cells had variable CCR4 expression. A proportion of IL-17+ synovial T cells produced IL-22 and interferon-γ. Conclusion This study is the first to define the frequency and characteristics of “Th17” cells in JIA. We suggest that these highly proinflammatory cells contribute to joint pathology, as indicated by relationships with clinical phenotypes, and that the balance between IL-17+ T cells and Treg cells may be critical to outcome.

Modulation of dendritic cell function by naive and regulatory CD4+ T cells.

The consequences of interactions between dendric cells (DCs) and either naive CD4+ T cells or regulatory CD4+CD25+ T cells on the expression of proinflammatory IL-6 and anti-inflammatory IL-10 in DC were examined over a period of 12 h, spanning the time frame during which stable T cell–DC interactions shape the development of tolerance and immunity in vivo. We demonstrate that the basal production of IL-6 and IL-10, which is initiated following DC stimulation with LPS, is modified in distinctly different ways by interaction with the two T cell populations. Naive CD4 T cells skew DC cytokine production toward IL-6 and suppress IL-10, whereas CD4+CD25+ T cells have the opposite effect. CD8 T cells or memory CD4 T cells do not influence basal cytokine production by stimulated DC. The effect of CD4+CD25+ T cells is dominant in coculture with naive CD4 T cells as long as inflammatory LPS is absent; the addition of LPS abrogates the suppression of IL-6. However, the modulating influence of CD4+CD25+ T cells remains evident in the enhancement of IL-10 production. Thus, mutual interactions between DC and CD4+ T cell subpopulations following contact with pathogens are likely to influence the strength and quality of incipient immune responses in the local microenvironment.

High expression of the ectonucleotidase CD39 on T cells from the inflamed site identifies two distinct populations, one regulatory and one memory T cell population.

The ectonucleotidase CD39 has recently been described as being highly expressed on regulatory Foxp3+ CD4 T cells. Through hydrolysis of proinflammatory extracellular ATP, CD39 activity represents a newly described mechanism of regulatory T cell action. We report a novel population of human CD4 T cells that express CD39 yet are Foxp3 negative. These cells produce the proinflammatory cytokines IFN-γ and IL-17 and fail to suppress proliferation; however, they still have high ATP hydrolysis activity. In the inflammatory site in human juvenile idiopathic arthritis, the CD39+Foxp3− population is greatly increased compared with peripheral blood of patients or healthy controls. We also show that cells expressing the AMPase CD73 are less frequent in the joint than in blood. To our knowledge, this is the first study to describe and characterize CD39 function on CD4 T cells from the target site in a human autoinflammatory condition. Our data suggest that in human CD4+ T cells from the inflamed site, CD39 can be highly expressed on two populations, one regulatory and the other of a memory phenotype.

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.

Overexpression of MHC Class I Heavy Chain Protein in Young Skeletal Muscle Leads to Severe Myositis : Implications for Juvenile Myositis

Folding and transport of proteins, such as major histocompatibility complex (MHC) class I, through the endoplasmic reticulum (ER) is tightly regulated in all cells, including muscle tissue, where the specialized ER sarcoplasmic reticulum is also critical to muscle fiber function. Overexpression of MHC class I protein is a common feature of many muscle pathologies including idiopathic myositis and can induce ER stress. However, there has been no comparison of the consequences of MHC overexpression in muscle at different ages. We have adapted a transgenic model of myositis induced by overexpression of MHC class I protein in skeletal muscle to investigate the effects of this protein overload on young muscle fibers, as compared with adult tissue. We find a markedly more severe disease phenotype in young mice, with rapid onset of muscle weakness and pathology. Gene expression profiling to compare the two models indicates rapid onset of ER stress in young muscle tissue but also that gene expression of key muscle structural proteins is affected more rapidly in young mice than adults after this insult. This novel model has important implications for our understanding of muscle pathology in dermatomyositis of both adults and children.

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.

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.