Wnt-1-inducible signaling pathway protein 3 and susceptibility to juvenile idiopathic arthritis

OBJECTIVE: To determine whether Wnt-1-inducible signaling pathway protein 3 (WISP3) polymorphisms are associated with susceptibility to juvenile idiopathic arthritis (JIA). METHODS: The exons and the intron/exon boundaries of the WISP3 gene were mutation-screened by denaturing high-performance liquid chromatography in 86 patients with polyarticular-course JIA (>/=5 joints affected) and 15 controls. Seven single-nucleotide polymorphisms (SNPs) were genotyped, using allelic discrimination, in a case-control study. Initially, 159 patients with polyarticular-course JIA and 263 controls were studied, followed by study of a replication cohort of 181 patients with polyarticular-course JIA and 355 controls. Available parents of patients with polyarticular-course JIA were also genotyped. Finally, other JIA subgroups were studied (initial cohort, n = 218; replication cohort, n = 213). Single-point and haplotype analysis was carried out. RESULTS: Positive association with SNP WISP3*G84A was observed and replicated in 2 cohorts of patients with polyarticular-course JIA. Specifically, homozygosity of the mutant allele (WISP3*84AA) conferred a 2-fold increased risk of disease susceptibility (for the initial cohort, odds ratio [OR] 2.1, 95% confidence interval [95% CI] 1.1-4.2, P = 0.03; for the replication cohort, OR 2.0, 95% CI 1.0-4.3, P = 0.05). Strong linkage disequilibrium was observed between SNPs; however, no haplotypic effect of an order of magnitude greater than the single-point WISP3*G84A association was observed. Using the transmission disequilibrium test, a trend toward overtransmission of the WISP3*84A allele was observed in patients with polyarticular-course JIA. No association of any WISP3 polymorphism was observed in the other JIA subgroups. CONCLUSION: Association and replication of a polymorphism within the first intron of the WISP3 gene have been shown in patients with polyarticular-course JIA. The functional significance of the WISP3*G84A SNP is being determined.     

Autoinflammatory genes and susceptibility to psoriatic juvenile idiopathic arthritis

OBJECTIVE: To investigate the association of NLRP3, NOD2, MEFV, and PSTPIP1, genes that cause 4 of the autoinflammatory hereditary periodic fever syndromes (HPFS), with juvenile idiopathic arthritis (JIA). METHODS: Fifty-one single-nucleotide polymorphisms (SNPs) across the 4 loci were investigated using MassArray genotyping in 950 Caucasian patients with JIA living in the UK and 728 ethnically matched healthy controls. RESULTS: Prior to Bonferroni correction for multiple testing, significant genotype associations between 6 SNPs in MEFV and JIA were observed and, in subgroup analysis, associations between 12 SNPs across all 4 loci and the subgroup of patients with psoriatic JIA were found. After Bonferroni correction for multiple testing, 2 genotype associations remained significant in the subgroup of patients with psoriatic JIA (MEFV SNP rs224204 [corrected P = 0.025] and NLRP3 SNP rs3806265 [corrected P = 0.04]). CONCLUSION: These findings support the use of monogenic loci as candidates for investigating the genetic component of complex disease and provide preliminary evidence of association between SNPs in autoinflammatory genes and psoriatic JIA. Our findings raise the interesting possibility of a shared disease mechanism between the HPFS and psoriatic JIA, potentially involving abnormal production of interleukin-1beta.     

Neuroendocrine gene polymorphisms and susceptibility to juvenile idiopathic arthritis

OBJECTIVE: To investigate the involvement of neuroendocrine candidate genes in the aetiopathogenesis of juvenile idiopathic arthritis (JIA). METHODS: Single-nucleotide polymorphisms and intragenic microsatellite markers within five neuroendocrine candidate genes (CRH, CBG, CYP19, ESR1, PRL) were investigated in 463 clinically characterized UK Caucasian JIA patients and a panel of 276 unrelated, healthy UK Caucasian controls. RESULTS: None of the polymorphisms investigated showed any statistically significant associations with JIA. CONCLUSIONS: The lack of association with polymorphisms of these neuroendocrine genes suggests that they are not involved in susceptibility to JIA.     

Cytokine gene polymorphisms and susceptibility to juvenile idiopathic arthritis

Objective

To investigate the involvement of candidate cytokine genes in the pathogenesis of juvenile idiopathic arthritis (JIA).

Methods

Single nucleotide polymorphisms and intragenic microsatellite markers within 8 candidate cytokine genes (interleukin‐1α [IL‐1α], IL‐2, IL‐4, IL‐6, IL‐10, interferon‐α1 [IFNA1], interferon‐γ [IFNG], and interferon regulatory factor 1 [IRF‐1]) were investigated in 417 Caucasian patients with clinically characterized JIA and a panel of 276 unrelated, healthy Caucasian controls, all from the United Kingdom.

Results

A novel 3′–untranslated region (3′UTR) polymorphism in IRF‐1 was found to be associated with susceptibility to JIA (corrected P = 0.002). No significant association with IL‐1α, IL‐2, IL‐4, IL‐6, IL‐10, IFNA1, or IFNG was observed.

Conclusion

An association between JIA and a previously unreported 3′UTR polymorphism of IRF‐1 was observed. This association was not found to be specific to any particular JIA subgroup. This suggests that IRF‐1 may contribute to a common pathogenesis shared by all JIA patients, regardless of clinical phenotype. This is most likely to be a genetic contribution to the chronic inflammatory process that underlies JIA pathology.

     

Wnt1 inducible signaling pathway protein-3 regulation and microsatellite structure in arthritis

OBJECTIVE: Rheumatoid arthritis (RA) synovial tissue expresses several embryonic gene families, including wingless (wnt) and their receptors, frizzled (fz). The Wnt proteins, including Wnt-1, activate the Wnt inducible signaling pathway proteins (WISP), which are members of the CCN family that regulate cell growth and differentiation. WISP3 is of particular interest because it contains a microsatellite region in its coding region that is susceptible to frameshift mutations and leads to a truncated protein. To investigate the contribution of WISP3 to synovial inflammation, we evaluated its expression and regulation in arthritis. METHODS: mRNA and protein expression of WISP3 were determined by quantitative real-time polymerase chain reaction (PCR) and Western blot analysis, respectively. For mutation analysis, PCR product amplified from genomic DNA of synovial tissue and cultured fibroblast-like synoviocytes (FLS) was subcloned and sequenced. RESULTS: WISP3 mRNA is expressed in synovial tissue, but is 11-fold higher in RA than osteoarthritis (OA) or normal samples. Surprisingly, WISP3 protein levels are similar in RA, OA, and normal synovium samples. Immunohistochemistry of synovial tissue reveals that WISP3 protein is located primarily in the synovial intimal lining. WISP3 mRNA expression is also 6-fold higher in RA FLS compared with OA FLS and 50-fold higher in RA than in normal FLS. When RA FLS are stimulated with interleukin 1 or tumor necrosis factor-a, WISP3 mRNA is significantly increased. The cytokines also increase WISP3 mRNA in OA FLS, but the maximal level in stimulated OA FLS is still less than medium-treated RA FLS. Mutation analysis in the coding region microsatellite of the WISP3 gene in RA and OA synovium and FLS shows a limited number of insertion and deletion mutations. CONCLUSION: WISP3 gene expression is higher in RA synovium and FLS compared with OA and normal synovial tissue and is further induced by proinflammatory cytokines in vitro. Protein levels are not increased, indicating discoordinate regulation of WISP3 protein and mRNA. Although functionally relevant mutations were observed in genomic DNA, they were noted in both OA and RA samples.     

GSTT1, GSTM1 and GSTP1 polymorphisms and susceptibility to juvenile idiopathic arthritis

OBJECTIVE: In this study we have analyzed GSTM1, GSTT1 and GSTP1 polymorphisms in patients with juvenile idiopathic arthritis (JIA), to investigate a possible role of these genes as genetic components of the disease. METHODS: A total of 103 individuals (49 oligoarticular, 41 polyarticular and 13 systemic) were analyzed for the three polymorphisms, using a PCR/RFLP methodology. RESULTS: We have observed significantly increased frequencies of individuals with GSTT1 null genotype in JIA patients comparing to controls (37% x 21%; p=0.0183). There was a 2-fold increased risk (OR 2.2, 95% CI 1.2-4.1) associating the disease with the GSTT1 null genotype. Considering the subgroups (oligoarticular, polyarticular and systemic), the results indicated an association between polyarticular and systemic patients and the GSTT1 null genotype. There was a 2-fold increased risk for polyarticular patients (OR 2.4, 95%, CI 1.1-5.4), and a 4-fold increased risk for systemic patients (OR 4.4, 95%, 1.3-14.5). CONCLUSION: The GSTT1 null genotype seems to be involved in polyarticular and systemic JIA.     

Involvement of the Wnt signaling pathway in experimental and human osteoarthritis: Prominent role of Wnt‐induced signaling protein 1

Objective

Wnt signaling pathway proteins are involved in embryonic development of cartilage and bone, and, interestingly, developmental processes appear to be recapitulated in osteoarthritic (OA) cartilage. The present study was undertaken to characterize the expression pattern of Wnt and Fz genes during experimental OA and to determine the function of selected genes in experimental and human OA.

Methods

Longitudinal expression analysis was performed in 2 models of OA. Levels of messenger RNA for genes from the Wnt/β‐catenin pathway were determined in synovium and cartilage, and the results were validated using immunohistochemistry. Effects of selected genes were assessed in vitro using recombinant protein, and in vivo by adenoviral overexpression.

Results

Wnt‐induced signaling protein 1 (WISP‐1) expression was strongly increased in the synovium and cartilage of mice with experimental OA. Wnt‐16 and Wnt‐2B were also markedly up‐regulated during the course of disease. Interestingly, increased WISP‐1 expression was also found in human OA cartilage and synovium. Stimulation of macrophages and chondrocytes with recombinant WISP‐1 resulted in interleukin‐1–independent induction of several matrix metalloproteinases (MMPs) and aggrecanase. Adenoviral overexpression of WISP‐1 in murine knee joints induced MMP and aggrecanase expression and resulted in cartilage damage.

Conclusion

This study included a comprehensive characterization of Wnt and Frizzled gene expression in experimental and human OA articular joint tissue. The data demonstrate, for the first time, that WISP‐1 expression is a feature of experimental and human OA and that WISP‐1 regulates chondrocyte and macrophage MMP and aggrecanase expression and is capable of inducing articular cartilage damage in models of OA.

     

Suppression of collagen‐induced arthritis in growth arrest and DNA damage–inducible protein 45β–deficient mice

Objective

Growth arrest and DNA damage–inducible protein 45β (GADD45β) is involved in stress responses, cell cycle regulation, and oncogenesis. Previous studies demonstrated that GADD45β deficiency exacerbates K/BxN serum–induced arthritis and experimental allergic encephalomyelitis (EAE) in mice, indicating that GADD45β plays a suppressive role in innate and adaptive immune responses. To further understand how GADD45β regulates autoimmunity, we evaluated collagen‐induced arthritis in GADD45β^–/– mice.

Methods

Wild‐type (WT) and GADD45β^–/– DBA/1 mice were immunized with bovine type II collagen (CII). Serum anticollagen antibody levels were quantified by enzyme‐linked immunosorbent assay. Expression of cytokines and matrix metalloproteinases in the joint and spleen was determined by quantitative polymerase chain reaction. The in vitro T cell cytokine response to CII was measured by multiplex analysis. CD4+CD25+ Treg cells and Th17 cells were quantified using flow cytometry.

Results

GADD45β^–/– mice showed significantly lower arthritis severity and joint destruction compared with WT mice. MMP‐3 and MMP‐13 expression was also markedly reduced in GADD45β^–/– mice. However, serum anti‐CII antibody levels were similar in both groups. FoxP3 and interleukin‐10 (IL‐10) expression was increased 2–3‐fold in splenocytes from arthritic GADD45β^–/– mice compared with those from WT mice. Flow cytometric analysis showed greater numbers of CD4+CD25+ Treg cells in the spleen of GADD45β^–/– mice than in the spleen of WT mice. In vitro studies showed that interferon‐γ and IL‐17 production by T cells was significantly decreased in GADD45β^–/– mice.

Conclusion

Unlike passive K/BxN arthritis and EAE, GADD45β deficiency in CIA was associated with lower arthritis severity, elevated IL‐10 expression, decreased IL‐17 production, and increased numbers of Treg cells. The data suggest that GADD45β plays a complex role in regulating adaptive immunity and, depending on the model, either enhances or suppresses inflammation.

     

Toll-like receptor 4 gene polymorphisms and susceptibility to juvenile idiopathic arthritis

OBJECTIVES: To determine if polymorphisms within the Toll-like receptor 4 (TLR4) gene are associated and linked with juvenile idiopathic arthritis (JIA). To investigate any possible gene-gene (epistatic) interaction between TLR4 and macrophage migration inhibitory factor (MIF) gene polymorphisms. METHODS: 313 simplex families (each containing one affected JIA proband) were genotyped. Two known functionally important single nucleotide polymorphisms (SNPs) within the TLR4 gene (Asp299Gly and Thr399Ile) were typed by SNaPshot ddNTP primer extension and capillary electrophoresis.Single point and multipoint transmission disequilibrium tests (TDT) were carried out through the extended TDT and TDT phase packages for the two TLR4 SNPs. Epistatic interaction between TLR4 haplotypes and the previously JIA associated MIF CATT(7)-MIF-173*C promoter haplotype was investigated by chi(2) test and unconditional logistic regression in Stata version 7. RESULTS: No distortion from random inheritance was observed by single point analysis for TLR4 Asp299Gly (p = 0.89) or TLR4 Thr399Ile (p = 0.40). Similarly, no distortion in transmission was seen when the TLR4 haplotypes were studied (p = 0.54). Additionally, no evidence for gene-gene interaction between TLR4 polymorphisms and the previously associated MIF gene polymorphisms was found (p = 0.40). CONCLUSIONS: No linkage or association was seen for Asp299Gly or Thr399Ile SNPs of TLR4 with JIA susceptibility. No evidence of an epistatic interaction between these TLR4 polymorphisms and MIF polymorphisms was found.     

Genetic loci contributing to hemophagocytic lymphohistiocytosis do not confer susceptibility to systemic‐onset juvenile idiopathic arthritis

Objective

To investigate whether single‐nucleotide polymorphisms (SNPs) within the genes PRF1, GZMB, UNC13D, and Rab27a, which are involved in natural killer cell dysfunction and known to contribute to the risk of hemophagocytic lymphohistiocytosis (HLH), confer an increased risk of susceptibility to systemic‐onset juvenile idiopathic arthritis (JIA).

Methods

Four SNPs across the PRF1 gene locus, 5 for GZMB, 7 for UNC13D, and 11 for Rab27a were investigated using MassArray genotyping in 133 UK Caucasian patients with systemic‐onset JIA and 384 ethnically matched unrelated control subjects. Additional control genotypes were accessed from the data generated by the Wellcome Trust Case Control Consortium.

Results

No significant association was found between any SNP within the 4 selected loci and systemic‐onset JIA, by either single‐point or haplotype analysis.

Conclusion

The results of this study demonstrate that genes involved in HLH do not confer a significant risk of association with systemic‐onset JIA.

     

Murine tumor necrosis factor α–induced adipose‐related protein (tumor necrosis factor α–induced protein 9) deficiency leads to arthritis via interleukin‐6 overproduction with enhanced NF‐κB,STAT‐3 signaling,and dysregulated apoptosis of macrophages

Objective

To elucidate the role of tumor necrosis factor α–induced adipose‐related protein (TIARP; or tumor necrosis factor α–induced protein 9 [TNFAIP‐9]) in the development and pathogenesis of arthritis.

Methods

We generated TIARP‐deficient (TIARP^−/−) mice and investigated several organs in aged mice. Peritoneal macrophages were collected and cultured with lipopolysaccharide (LPS) and TNFα, and then the production of cytokines and subsequent NF‐κB signal transduction were analyzed. We also examined the susceptibility of young TIARP^−/− mice to collagen‐induced arthritis (CIA). Draining lymph nodes and splenocytes were isolated and cultured, and serum levels of anti–type II collagen (anti‐CII) antibodies, interleukin‐6 (IL‐6), and TNFα on day 60 were measured. We further investigated the effects of anti–IL‐6 receptor monoclonal antibody (mAb) on the development of arthritis in TIARP^−/− mice. IL‐6/STAT‐3 signaling was also analyzed using TIARP^−/− macrophages.

Results

TIARP^−/− mice developed spontaneous enthesitis and synovitis, had high serum levels of IL‐6, had increased CD11b+ cell counts in the spleen, and showed enhanced LPS‐ and TNFα‐induced IL‐6 expression in macrophages. Sustained degradation of IκBα with dysregulated apoptosis was also noted in TIARP^−/− macrophages. CIA was clearly exacerbated in TIARP^−/− mice, accompanied by marked neutrophil and macrophage infiltration in joints. The levels of anti‐CII antibodies in serum were unchanged, whereas autoreactive Th1 cell and Th17 cell responses were higher in TIARP^−/− mice. Treatment with anti–IL‐6 receptor mAb prevented the development of CIA in TIARP^−/− mice, and TIARP^−/− macrophages showed increased IL‐6–induced STAT‐3 phosphorylation.

Conclusion

These findings suggest that TIARP acts as a negative regulator of arthritis by suppressing IL‐6 production, its signaling and TNFα‐induced NF‐κB signaling, resulting in enhanced apoptosis in macrophages.

     

Positive association of SLC26A2 gene polymorphisms with susceptibility to systemic‐onset juvenile idiopathic arthritis

Objective

To investigate SLC26A2, the gene that causes diastrophic dysplasia, in juvenile idiopathic arthritis (JIA).

Methods

Nine polymorphisms across the SLC26A2 gene locus were investigated using MassArray genotyping in 826 UK Caucasian JIA cases and 617 ethnically matched healthy controls.

Results

Significant associations between multiple single‐nucleotide polymorphisms (SNPs) across SLC26A2 and systemic‐onset JIA were found. In each case, homozygosity for the minor allele conferred the increased risk of disease susceptibility: rs1541915 (odds ratio [OR] 2.3, 95% confidence interval [95% CI] 1.4–3.7, P = 0.0003), rs245056 (OR 2.8, 95% CI 1.7–4.6, P = 0.00002), rs245055 (OR 2.5, 95% CI 1.2–5.0, P = 0.004), rs245051 (OR 2.3, 95% CI 1.4–3.7, P = 0.0005), rs245076 (OR 2.7, 95% CI 1.3–5.4, P = 0.0015), and rs8073 (OR 2.3, 95% CI 0.9–5.6, P = 0.04).

Conclusion

These findings show the value of using monogenic disease loci as candidates for investigation in JIA. We identified a subgroup‐specific association between SNPs within the SLC26A2 gene and systemic‐onset JIA. Our findings also highlight systemic‐onset JIA as being a distinctly different disease from that in the other JIA subgroups.

     

Interferon‐α–inducible proteins are novel autoantigens in murine lupus

Objective

To investigate the spectrum of B cell autoimmunity in the recently described anti‐CD1–autoreactive T cell receptor (TCR)–transgenic murine lupus‐like (CD1 lupus‐like) model.

Methods

Lethally irradiated BALB/c/nu/nu mice were injected intravenously with donor BALB/c bone marrow and spleen cells expressing TCRα and TCRβ transgenes that recognize CD1d. Sera from adoptive host animals that developed lupus (i.e., CD1 lupus mice) were collected at serial time points and analyzed by Western blotting and immunoprecipitation, using protein extracts prepared from NIH3T3 mouse fibroblasts and EL‐4 lymphocytes, respectively. Sera obtained from older animals in several models of spontaneous lupus (NZB/NZW, MRL++, and MRL/lpr mice), unmanipulated BALB/c/nu/nu mice, and normal BALB/c mice were used as controls.

Results

Analyses demonstrated that the prominent targets of autoantibodies in the CD1 lupus‐like model are interferon‐α (IFNα)–inducible antigens. Biochemical and serologic characterizations identified one antigen as belonging to the interferon‐inducible 202 (Ifi202) subfamily of proteins within the Ifi200 family, and a second antigen as a member of the 70‐kd heat‐shock protein family. Autoantibodies directed against these antigens were rapidly produced at an early stage of disease. Anti‐p50 autoantibodies were present in sera from 7 (78%) of 9 CD1 lupus mice that developed severe kidney disease.

Conclusion

IFNα‐inducible proteins represent a novel class of autoantigens in murine lupus, and the findings suggest additional roles for IFNα in this disease. Since Ifi202 autoantigens are encoded by the murine non–major histocompatibility complex lupus‐susceptibility gene locus Ifi202, these data provide a link between recent advances in lupus genetics and the formation of autoantibodies.

     

Clinical outcomes after withdrawal of anti–tumor necrosis factor α therapy in patients with juvenile idiopathic arthritis: A twelve‐year experience

Objective

To estimate the length of time to disease flare and the likelihood of achieving clinical remission after discontinuation of treatment with tumor necrosis factor α (TNFα) blockers in patients with juvenile idiopathic arthritis (JIA).

Methods

We conducted a retrospective chart review in a cohort of patients with JIA treated with TNFα inhibitors between January 1, 1998 and November 1, 2009. Demographic information, laboratory data, and medication exposure were extracted using a standardized tool. Outcomes of interest were based on preliminary criteria for remission in JIA.

Results

One hundred seventy‐one patients with 255 discrete episodes of anti‐TNFα treatment were reviewed. The median duration of patient observation was 59.7 months (range 5.8–211.2 months). Among patients in whom disease was inactive after discontinuation of anti‐TNFα therapy, 50% had persistently inactive disease at 6 months, and 33% had clinical remission at 12 months. The median duration of anti‐TNFα therapy after inactive disease was obtained was 6.1 months (range 0–67.9 months). No significant association was observed between the time to disease flare after cessation of treatment with TNFα antagonists and the length of time from the diagnosis of JIA to the initiation of anti‐TNFα therapy, the duration of therapy following the onset of inactive disease, or the total duration of treatment with TNFα antagonists prior to discontinuation. The category of JIA, sex, and age at diagnosis were not associated with the risk of relapse.

Conclusion

One‐third of patients with JIA can successfully undergo withdrawal of treatment with TNFα antagonists and be spared the cost and potential morbidity of treatment for at least 12 months. Further studies are needed to identify factors to accurately identify these patients.