Development of Monitoring System for Assessing Rheumatoid Arthritis within 5 Minutes Using a Drop of Bio-Fluids

Rheumatoid arthritis (RA) disease activity fluctuates over time. The disease activity score 28 (DAS28ESR) is a widely used and validated scoring system for assessing RA activity; however, it requires time and expertise. This study aimed to develop a new molecular assay capable of rapidly and objectively assessing RA activity. We used a rapid immuno-assay system (FREND™) to measure soluble CD14 (sCD14) levels, which reflect the DAS28ESR. SCD14 concentrations in urine and serum of RA patients were measured, and RA activity and responses to anti-rheumatic drugs were examined at baseline and after 6 months. FREND™ quantified sCD14 levels in a drop of serum and urine accurately and within 5 min. Serum sCD14 concentrations and its changes correlated well with disease activity and treatment responses, and the results were comparable to C-reactive protein. The new composite indices, including the DAS28CD14 and simplified DASCD14, better detected RA activity than a single sCD14 value and correlated strongly with the DAS28ESR. These indices exhibited excellent diagnostic performance for discriminating a good response 6 months after treatment. We developed a new system for assessing RA activity and therapeutic outcome within 5 min. CD14-based composite indices may have utility for accurate and frequent monitoring of RA status

Transcription Factor NFAT5 Promotes Migration and Invasion of Rheumatoid Synoviocytes via Coagulation Factor III and CCL2

Fibroblast-like synoviocytes (FLSs) play a key role in the progression of rheumatoid arthritis (RA) as a primary component of invasive hypertrophied pannus. FLSs of RA patients (RA-FLSs) exhibit cancer-like features, including promigratory and proinvasive activities that largely contribute to joint cartilage and bone destruction. In this study, we hypothesized that the NF of activated T cell 5 (NFAT5), a transcription factor involving tumor invasiveness, would control the migration and invasion of RA-FLSs. Analyses of transcriptomes demonstrated the significant involvement of NFAT5 in locomotion of RA-FLSs and that tissue factor (TF; also known as coagulation factor III) and CCL2 were the major downstream target genes of NFAT5 involving FLS migration and invasion. In cultured RA-FLSs, IL-1 beta and TGF-beta increased TF and CCL2 expression by upregulating NFAT5 expression via p38 MAPK. Functional assays demonstrated that NFAT5-or TF-deficient RA-FLSs displayed decreased lamellipodia formation, cell migration, and invasion under IL-1 beta- or TGF-beta-stimulated conditions. Conversely, factor VIIa, a specific activator of TF, increased migration of RA-FLSs, which was blocked by NFAT5 knockdown. Recombinant CCL2 partially restored the decrease in migration and invasion of NFAT5-deficient RA-FLSs stimulated with IL-1 beta. NFAT5-knockout mouse FLSs also showed decreased expressions of TF and CCL2 and reduced cell migration. Moreover, KRN2, a specific inhibitor of NFAT5, suppressed migration of FLSs stimulated with TGF-beta. Conclusively, to our knowledge, this is the first study to provide evidence of a functional link between osmoprotective NFAT5 and TF in the migration and invasion of RA-FLSs and supports a role for NFAT5 blockade in the treatment of RA

Transcriptional Regulator CTR9 Inhibits Th17 Differentiation via Repression of IL-17 Expression.

PAF complex is an evolutionarily conserved transcriptional complex that associates with RNA polymerase II in the coding region of actively transcribing genes. Although its transcriptional activity is closely related to diverse cellular processes, such as cell-cycle progression or development in mammals, its role in immune responses has not been addressed yet. In this study, we show that CTR9, a component of PAF complex, functions as a repressor of Th17 differentiation. Both mRNA and protein levels of CTR9 were significantly decreased during the differentiation processes of naive T into Th17 effector cells. When CTR9 was depleted, IL-17 expression was induced and differentiation into Th17 cells enhanced. In naive T cells, CTR9 occupied the coding region of Il17a, but dissociated under Th17 in vitro-polarizing conditions. In contrast, both CDC73 and PAF1 were recruited to the Il17a locus under Th17-differentiation conditions. In the IL-6-stimulated splenocytes, expression of CTR9 was decreased, and chromatin-bound CTR9 disappeared in the coding region of Il17a. IL-6 also directly repressed expression of CTR9 gene, as promoter activity of CTR9 was similarly repressed by IL-6 treatment. Moreover, in mice with collagen-induced arthritis, lentivirus-mediated CTR9 overexpression in the joints ameliorated arthritis severity, decreasing the frequency of CD4(+) IL-17(+) T cells in lymph nodes. In conclusion, our data propose a novel feed-forward loop of IL-17 transcriptional regulatory circuit, via IL-6-mediated repression of CTR9 which is a transcriptional repressor of IL-17.X1144sciescopu

Leukocyte-specific protein 1 regulates T-cell migration in rheumatoid arthritis

Copy number variations (CNVs) have been implicated in human diseases. However, it remains unclear how they affect immune dysfunction and autoimmune diseases, including rheumatoid arthritis (RA). Here, we identified a novel leukocyte-specific protein 1 (LSP1) deletion variant for RA susceptibility located in 11p15.5. We replicated that the copy number of LSP1 gene is significantly lower in patients with RA, which correlates positively with LSP1 protein expression levels. Differentially expressed genes in Lsp1-deficient primary T cells represent cell motility and immune and cytokine responses. Functional assays demonstrated that LSP1, induced by T-cell receptor activation, negatively regulates T-cell migration by reducing ERK activation in vitro. In mice with T-cell-dependent chronic inflammation, loss of Lsp1 promotes migration of T cells into the target tissues as well as draining lymph nodes, exacerbating disease severity. Moreover, patients with RA show diminished expression of LSP1 in peripheral T cells with increased migratory capacity, suggesting that the defect in LSP1 signaling lowers the threshold for T-cell activation. To our knowledge, our work is the first to demonstrate how CNVs result in immune dysfunction and a disease phenotype. Particularly, our data highlight the importance of LSP1 CNVs and LSP1 insufficiency in the pathogenesis of RA and provide previously unidentified insights into the mechanisms underlying T-cell migration toward the inflamed synovium in RA.