Lorie Luyrink

Subtype-specific peripheral blood gene expression profiles in recent onset juvenile idiopathic arthritis

OBJECTIVE To identify differences in peripheral blood gene expression between patients with different subclasses of juvenile idiopathic arthritis (JIA) and healthy controls in a multicenter study of patients with recent-onset JIA prior to treatment with disease-modifying antirheumatic drugs (DMARDs) or biologic agents. METHODS Peripheral blood mononuclear cells (PBMCs) from 59 healthy children and 136 patients with JIA (28 with enthesitis-related arthritis [ERA], 42 with persistent oligoarthritis, 45 with rheumatoid factor [RF]-negative polyarthritis, and 21 with systemic disease) were isolated from whole blood. Poly(A) RNA was labeled using a commercial RNA amplification and labeling system (NuGEN Ovation), and gene expression profiles were obtained using commercial expression microarrays (Affymetrix HG-U133 Plus 2.0). RESULTS A total of 9,501 differentially expressed probe sets were identified among the JIA subtypes and controls (by analysis of variance; false discovery rate 5%). Specifically, 193, 1,036, 873, and 7,595 probe sets were different in PBMCs from the controls compared with those from the ERA, persistent oligoarthritis, RF-negative polyarthritis, and systemic JIA patients, respectively. In patients with persistent oligoarthritis, RF-negative polyarthritis, and systemic JIA subtypes, up-regulation of genes associated with interleukin-10 (IL-10) signaling was prominent. A hemoglobin cluster was identified that was underexpressed in ERA patients but overexpressed in systemic JIA patients. The influence of JAK/STAT, ERK/MAPK, IL-2, and B cell receptor signaling pathways was evident in patients with persistent oligoarthritis. In systemic JIA, up-regulation of innate immune pathways, including IL-6, Toll-like receptor/IL-1 receptor, and peroxisome proliferator-activated receptor signaling, were noted, along with down-regulation of gene networks related to natural killer cells and T cells. Complement and coagulation pathways were up-regulated in systemic JIA, with a subset of these genes being differentially expressed in other subtypes as well. CONCLUSION Expression analysis identified differentially expressed genes in PBMCs obtained early in the disease from patients with different subtypes of JIA and in healthy controls, providing evidence of immunobiologic differences between these forms of childhood arthritis.

Polymorphism in a T-cell receptor variable gene is associated with susceptibility to a juvenile rheumatoid arthritis subset

This report demonstrates a T-cell receptor (Tcr) restriction fragment length polymorphism, defined by a Tcrb-V6.1 gene probe and Bgl II restriction enzyme, to be absolutely correlated with allelic variation in the coding sequence of a Tcrb-V6.1 gene. A pair of non-conservative amino acid substitutions distinguish the Tcrb-V6.1 allelic variants. An association of this Tcrb-V6.1 gene allelic variant with one form of juvenile rheumatoid arthritis (JRA) was established in a cohort of 126 patients. The association was observed in patients possessing the HLA-DQA1*0101 gene. Among HLA-DQA*0101 individuals, 19 of 26 patients (73.1%) carried one particular Tcrb-V6.1 gene allele as opposed to 11 of 33 controls (33%; p<0.005). Haplotypes carrying this HLA gene have previously been shown to confer increased risk for progression of arthritis in JRA. This demonstration of a disease-associated Tcrb-V gene allelic variant has not, to our knowledge, been previously reported and supports the contribution of polymorphism in the Tcr variable region genomic repertoire to human autoimmune disease.

Chemokine Receptor CCR4 on CD4+ T Cells in Juvenile Rheumatoid Arthritis Synovial Fluid Defines a Subset of Cells with Increased IL-4:IFN-γ mRNA Ratios

To understand the mechanisms that promote recruitment and survival of T cells within the pediatric inflamed joint, we have studied the expression of CCR4 and CCR5 on synovial fluid T cells and matched peripheral blood samples from juvenile rheumatoid arthritis (JRA) patients using three-color flow cytometric analysis. Thymus- and activation-regulated chemokine and macrophage-derived chemokine, ligands for CCR4, were measured by ELISA in JRA synovial fluid, JRA plasma, adult rheumatoid arthritis synovial fluid, and normal plasma. IL-4 and IFN-γ mRNA production was assessed in CD4+/CCR4+ and CD4+/CCR4− cell subsets. We found accumulations of both CCR4+ and CCR5+ T cells in JRA synovial fluids and a correlation for increased numbers of CCR4+ T cells in samples collected early in the disease process. Thymus- and activation-regulated chemokine was detected in JRA synovial fluid and plasma samples, but not in adult rheumatoid arthritis synovial fluid or control plasma. Macrophage-derived chemokine was present in all samples. CD4+/CCR4+ synovial lymphocytes produced more IL-4 and less IFN-γ than CD4+/CCR4− cells. These findings suggest that CCR4+ T cells in the JRA joint may function early in disease in an anti-inflammatory capacity through the production of type 2 cytokines and may play a role in determining disease phenotype.

Biologic similarities based on age at onset in oligoarticular and polyarticular subtypes of juvenile idiopathic arthritis.

OBJECTIVE To explore biologic correlates to age at onset in patients with juvenile idiopathic arthritis (JIA) using peripheral blood mononuclear cell (PBMC) gene expression analysis. METHODS PBMCs were isolated from 56 healthy controls and 104 patients with recent-onset JIA (39 with persistent oligoarticular JIA, 45 with rheumatoid factor-negative polyarticular JIA, and 20 with systemic JIA). RNA was amplified and labeled using NuGEN Ovation, and gene expression was assessed with Affymetrix HG-U133 Plus 2.0 GeneChips. RESULTS A total of 832 probe sets revealed gene expression differences (false discovery rate 5%) in PBMCs from children with oligoarticular JIA whose disease began before age 6 years (early-onset disease) compared with those whose disease began at or after age 6 years (late-onset disease). In patients with early-onset disease, there was greater expression of genes related to B cells and less expression of genes related to cells of the myeloid lineage. Support vector machine analyses identified samples from patients with early- or late-onset oligoarticular JIA (with 97% accuracy) or from patients with early- or late-onset polyarticular JIA (with 89% accuracy), but not from patients with systemic JIA or healthy controls. Principal components analysis showed that age at onset was the major classifier of samples from patients with oligoarticular JIA and patients with polyarticular JIA. CONCLUSION PBMC gene expression analysis reveals biologic differences between patients with early-and late-onset JIA, independent of classification based on the number of joints involved. These data suggest that age at onset may be an important parameter to consider in JIA classification. Furthermore, pathologic mechanisms may vary with age at onset, and understanding these processes may lead to improved treatment of JIA.

HLA-DP/DR interaction in children with juvenile rheumatoid arthritis

Among the major histocompatibility complex class II genes, HLA-DP is least understood in terms of its contributions to the normal immune response and to autoimmunity. The few autoimmune diseases with HLA-DP associations can provide insight into the function of HLADP. Among these diseases is one form of juvenile rheumatoid arthritis (JRA) which has been associated with HLA-DPw2 (Hoffman et al. 1986; Odum et al. 1986). This particular type of JRA, early-onset pauciarticular disease (EOPA-JRA), has also been associated with other HLA class II genes -primari ly HLA-DR5 and HLA-DRw8. The two other HLA-DR genes carried on HLA-DRw52 haplotypes, HLA-DR3 and HLA-DRw6, have been associated with disease in certain study populations (Albert et al. 1989). In this study, HLA-DP typing by restriction fragment length polymorphism (RFLP) was used to analyze which subtype of HLA-DPw2 predisposes to EOPA-JRA. Second, the question was addressed as to whether the HLADPw2 association with EOPA-JRA is dependent on the combined presence of other disease-associated HLA-DR genes, and if so, whether all the disease-predisposing HLA-DR genes interact with HLA-DPw2 in a similar way. The 97 Caucasian patients studied met the Americn Rheumatism Association criteria for pauciarticular JRA (Cassidy et al. 1986) and had an onset of disease prior to the age of 6 years. The 130 controls were ethnically similar to the disease population; 65 % of the patients and 53 % of the controls originated from the British Isles and Germany. Most individuals had previously been serologically HLA-DR typed (Van Kerckhove et al. 1988). To HLA-

A distinct HLA-DRw8 haplotype characterizes patients with juvenile rheumatoid arthritis.

We studied the first domain of the HLA-DRB1, HLA-DQA1, and HLA-DQB1 loci of 67 HLA-DRw8-positive Caucasians including 43 with early-onset pauciarticular juvenile rheumatoid arthritis (EOPA-JRA, alternatively known as early-onset pauciarticular juvenile chronic arthritis). Serology, restriction fragment length polymorphism (RFLP), and polymerase chain reaction (PCR) oligotyping revealed that 62, including all the EOPA-JRA patients, carried the HLA-DRB1*0801, DQA1*0401, DQB1*0402 genotype. Approximately onefifth of the controls carried atypical HLA-DRB1, HLA-DQA1, and/or HLA-DQB1 loci on their HLA-DRw8 haplotype confirmed by family studies. DNA sequences of HLA-DRB1, DQA1, and DQB1 alleles in patients and controls were identical to those previously reported. Disease association studies in 113 EOPA-JRA patients and 207 controls unselected for HLA-DRw8 revealed that the HLA-DRB1*0801, DQA1*0401, DQB1*0402 genotype was associated with a higher relative risk (RR) for disease (RR = 12.8, χ2 = 48.8, P < 10−4) than was the serologically defined presence of HLA-DRw8 (RR = 8, χ2 = 39, P < 10−4). Further analysis suggested that the DQ genes on HLA-DRw8 haplotypes are as likely as the DR genes to contribute to the pathogenesis of EOPA-JRA. This study increases to five the number of HLA-DR/DQ haplotypes identified in HLA-DRw8 Caucasians.

Polymorphic markers related to a single Tcrb-V6 gene segment

The central role of the antigen-specific alpha/beta T-cell receptor (Tcr) in immune recognition has led to a search for Tcr gene polymorphism relevant to autoimmune diseases. Previous reports primarily emphasized associations with constant (C)-region (Millward et al. 1987; Demaine et al. 1989; Freimark et al. 1987) restriction fragment lenght polymorphisms (RFLPs) as opposed to polymorphisms of variable (10-region genes, which determine the specificity of antigen-MHC recognition by the Tcr. Although Tcr-Vand C-region genes are linked, recent family studies have reported a lack of linkage disequilibrium between Vand C-region polymorphisms (Robinson and Kindt, 1987; Charmley et al. 1988; Charmley et al. 1990) indicating that C-region polymorphisms alone may be of limited value in studying Tcr disease association (Nivens et al. 1990; Charmley et al. 1990). Further study of disease associations has been hampered by the paucity of data regarding the extent of polymorphism of Tcr-V-region genes in normal Caucasian populations. In the present study, we evaluated RFLPs related to Tcrb-V genes in 100 normal, unrelated, Caucasoid individuals using five Tcrb-V gene specific cDNA probes, V4, V5, V6.1, V8.1 and V18 (Leiden and Strominger 1986; Yanagi et al. 1984). Southern blot analysis of genomic DNA digested with the restriction enzymes Bgl-II, Bam HI, Eco RI, and Taq I, was carried out as described (Southern 1975). Polymorphic restriction enzyme sites were detected by two restriction enzymes, Taq I and Bgl II, with the TcrbV6.1 cDNA probe. Each probe/enzyme combination defines a bi-allelic polymorphism. Hybridization of the Tcrb-V6.1 probe to blots containing Bgl II digested DNA revealed a variant band of 5.7 kilobases (kb) whose inten-

Transcriptional profiles of JIA patient blood with subsequent poor response to methotrexate

Objective The mechanisms that determine the efficacy or inefficacy of MTX in JIA are ill-defined. The objective of this study was to identify a gene expression transcriptional signature associated with poor response to MTX in patients with JIA. Methods RNA sequencing was used to measure gene expression in peripheral blood mononuclear cells collected from 47 patients with JIA prior to MTX treatment and 14 age-matched controls. Differentially expressed baseline genes between responders and non-responders were evaluated. Biological differences between all JIA patients and controls were explored by constructing a signature of differentially expressed genes. Unsupervised clustering and pathway analysis was performed. Results A signature of 99 differentially expressed genes (Bonferroni-corrected P < 0.05) capturing the biological differences between all JIA patients and controls was identified. Unsupervised clustering of samples based on this list of 99 genes produced subgroups enriched for MTX response status. Comparing this gene signature with reference signatures from sorted cell populations revealed high concordance between the expression signatures of monocytes and of MTX non-responders. CXCL8 (IL-8) was the most significantly differentially expressed gene transcript comparing all JIA patients with controls (Bonferroni-corrected P = 4.12 × 10-10). Conclusion Variability in clinical response to MTX in JIA patients is associated with differences in gene transcripts modulated in monocytes. These gene expression profiles may provide a basis for biomarkers predictive of treatment response.