DUSP6 deletion protects mice and reduces disease severity in autoimmune arthritis.

Receptor tyrosine kinases (RTKs) have an important role in arthritis severity and in models of rheumatoid arthritis (RA), but their regulation is not fully understood. The dual specificity phosphatase 6 (DUSP6) has been implicated in the regulation of RTK signaling, but never in the context of arthritis and autoimmunity. We used the KRN serum-induced arthritis (KSIA) model of RA and showed that DUSP6-/- mice were protected and had a 50% lower maximum arthritis score (p = 0.006) and reduced joint damage than C57BL/6 DUSP6+/+ controls. Serum levels of interleukin (IL) 10 were significantly increased (>2-fold), and IL6 decreased in DUSP6-/- mice. DUSP6-/- mice had increased numbers of IL10+ cells including Tr1 regulatory cells (p < 0.01). Introduction of the IL10-/- into DUSP6-/- (double knockout [KO]) reversed the DUSP6-/- protection. In conclusion, this study reports a pro-arthritic role for DUSP6. This discovery has the potential to generate a previously unknown target for therapies for RA and inflammatory diseases.

KIF1C and new Huntingtin-interacting protein 1 binding proteins regulate rheumatoid arthritis fibroblast-like synoviocytes' phenotypes.

Background: Huntingtin-interacting protein-1 (HIP1) is a new arthritis severity gene implicated in the regulation of the invasive properties of rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS). These invasive properties of FLS strongly correlate with radiographic and histology damage in patients with RA and rodent models of arthritis. While HIP1 has several intracellular functions, little is known about its binding proteins, and identifying them has the potential to expand our understanding of its role in cell invasion and other disease-contributing phenotypes, and potentially identify new targets for therapy. Methods: FLS cell lines from arthritic DA (highly invasive) and from arthritis-protected congenic rats R6 (minimally invasive), which differ in an amino-acid changing HIP1 SNP, were cultured and lysed, and proteins were immunoprecipitated with an anti-HIP1 antibody. Immunoprecipitates were analyzed by mass spectrometry. Differentially detected (bound) proteins were selected for functional experiments using siRNA knockdown in human RA FLS to examine their effect in cell invasiveness, adhesion, cell migration and proliferation, and immunofluorescence microscopy. Results: Proteins detected included a few known HIP1-binding proteins and several new ones. Forty-five proteins differed in levels detected in the DA versus R6 congenic mass spectrometry analyses. Thirty-two of these proteins were knocked down and studied in vitro, with 10 inducing significant changes in RA FLS phenotypes. Specifically, knockdown of five HIP1-binding protein genes (CHMP4BL1, COPE, KIF1C, YWHAG, and YWHAH) significantly decreased FLS invasiveness. Knockdown of KIF1C also reduced RA FLS migration. The binding of four selected proteins to human HIP1 was confirmed. KIF1C colocalized with lamellipodia, and its knockdown prevented RA FLS from developing an elongated morphology with thick linearized actin fibers or forming polarized lamellipodia, all required for cell mobility and invasion. Unlike HIP1, KIF1C knockdown did not affect Rac1 signaling. Conclusion: We have identified new HIP1-binding proteins and demonstrate that 10 of them regulate key FLS phenotypes. These HIP1-binding proteins have the potential to become new therapeutic targets and help better understand the RA FLS pathogenic behavior. KIF1C knockdown recapitulated the morphologic changes previously seen in the absence of HIP1, but did not affect the same cell signaling pathway, suggesting involvement in the regulation of different processes.

Cell-Specific Gene Networks and Drivers in Rheumatoid Arthritis Synovial Tissues.

Rheumatoid arthritis (RA) is a common autoimmune and inflammatory disease characterized by inflammation and hyperplasia of the synovial tissues. RA pathogenesis involves multiple cell types, genes, transcription factors (TFs) and networks. Yet, little is known about the TFs, and key drivers and networks regulating cell function and disease at the synovial tissue level, which is the site of disease. In the present study, we used available RNA-seq databases generated from synovial tissues and developed a novel approach to elucidate cell type-specific regulatory networks on synovial tissue genes in RA. We leverage established computational methodologies to infer sample-specific gene regulatory networks and applied statistical methods to compare network properties across phenotypic groups (RA versus osteoarthritis). We developed computational approaches to rank TFs based on their contribution to the observed phenotypic differences between RA and controls across different cell types. We identified 18,16,19,11 key regulators of fibroblast-like synoviocyte (FLS), T cells, B cells, and monocyte signatures and networks, respectively, in RA synovial tissues. Interestingly, FLS and B cells were driven by multiple independent co-regulatory TF clusters that included MITF, HLX, BACH1 (FLS) and KLF13, FOSB, FOSL1 (synovial B cells). However, monocytes were collectively governed by a single cluster of TF drivers, responsible for the main phenotypic differences between RA and controls, which included RFX5, IRF9, CREB5. Among several cell subset and pathway changes, we also detected reduced presence of NKT cell and eosinophils in RA synovial tissues. Overall, our novel approach identified new and previously unsuspected KDG, TF and networks and should help better understanding individual cell regulation and co-regulatory networks in RA pathogenesis, as well as potentially generate new targets for treatment.

Gene Network Analyses Identify Co-regulated Transcription Factors and BACH1 as a Key Driver in Rheumatoid Arthritis Fibroblast-like Synoviocytes.

RNA-sequencing and differential gene expression studies have significantly advanced our understanding of pathogenic pathways underlying Rheumatoid Arthritis (RA). Yet, little is known about cell-specific regulatory networks and their contributions to disease. In this study, we focused on fibroblast-like synoviocytes (FLS), a cell type central to disease pathogenesis and joint damage in RA. We used a strategy that computed sample-specific gene regulatory networks (GRNs) to compare network properties between RA and osteoarthritis FLS. We identified 28 transcription factors (TFs) as key regulators central to the signatures of RA FLS. Six of these TFs are new and have not been previously implicated in RA, and included BACH1, HLX, and TGIF1. Several of these TFs were found to be co-regulated, and BACH1 emerged as the most significant TF and regulator. The main BACH1 targets included those implicated in fatty acid metabolism and ferroptosis. The discovery of BACH1 was validated in experiments with RA FLS. Knockdown of BACH1 in RA FLS significantly affected the gene expression signatures, reduced cell adhesion and mobility, interfered with the formation of thick actin fibers, and prevented the polarized formation of lamellipodia, all required for the RA destructive behavior of FLS. This is the first time that BACH1 is shown to have a central role in the regulation of FLS phenotypes, and gene expression signatures, as well as in ferroptosis and fatty acid metabolism. These new discoveries have the potential to become new targets for treatments aimed at selectively targeting the RA FLS.

Cell-specific gene networks and drivers in rheumatoid arthritis synovial tissues.

Rheumatoid arthritis (RA) is a common autoimmune and inflammatory disease characterized by inflammation and hyperplasia of the synovial tissues. RA pathogenesis involves multiple cell types, genes, transcription factors (TFs) and networks. Yet, little is known about the TFs, and key drivers and networks regulating cell function and disease at the synovial tissue level, which is the site of disease. In the present study, we used available RNA-seq databases generated from synovial tissues and developed a novel approach to elucidate cell type-specific regulatory networks on synovial tissue genes in RA. We leverage established computational methodologies to infer sample-specific gene regulatory networks and applied statistical methods to compare network properties across phenotypic groups (RA versus osteoarthritis). We developed computational approaches to rank TFs based on their contribution to the observed phenotypic differences between RA and controls across different cell types. We identified 18 (fibroblast-like synoviocyte), 16 (T cells), 19 (B cells) and 11 (monocyte) key regulators in RA synovial tissues. Interestingly, fibroblast-like synoviocyte (FLS) and B cells were driven by multiple independent co-regulatory TF clusters that included MITF, HLX, BACH1 (FLS) and KLF13, FOSB, FOSL1 (B cells). However, monocytes were collectively governed by a single cluster of TF drivers, responsible for the main phenotypic differences between RA and controls, which included RFX5, IRF9, CREB5. Among several cell subset and pathway changes, we also detected reduced presence of Natural killer T (NKT) cells and eosinophils in RA synovial tissues. Overall, our novel approach identified new and previously unsuspected Key driver genes (KDG), TF and networks and should help better understanding individual cell regulation and co-regulatory networks in RA pathogenesis, as well as potentially generate new targets for treatment.

Practice patterns in the use of supplemental folic acid in rheumatoid arthritis patients treated with methotrexate

Objetivo: Determinar na prática nao só os padroes de uso de suplementaçao com ácido fólico (FA) em pacientes com artrite reumatóide (RA) tratados com metotrexato (MTX) em nosso centro médico, como também o tempo de permanência em uso e toxicidade da droga entre diferentes grupos de pacientes. Métodos: Foi constituído um grupo de 40 pacientes com RA que iniciaram MTX logo após estarem disponíveis os resultados do primeiro estudo de suplementaçao com ácido fólico. Foram obtidos de todos os pacientes, durante três anos, os dados clínico/demográficos e, anualmente, a administraçao e toxicidade acumulada de MTX/FA. Foram examinadas estatísticas descritivas e curvas de sobrevida. Resultados: Três padroes surgiram na prática: pacientes que receberam MTX desde o início (FAO n = 18), aqueles que receberam-no às vezes durante o estudo por causa da toxicidade (FAD n=8) e aqueles que nunca o receberam (FAN n = 14). O tempo global de permanência em uso da droga durante três anos foi de 69 por cento, sendo comparável nos três grupos. Os valores basais MCV (volume corpuscular médio) e os índices de toxicidade acumulada foram maiores entre os pacientes do grupo FAD. Conclusao: Nao foi universal a implementaçao de alteraçoes nas práticas terapêuticas entre nossos clínicos. A permanência em uso da droga foi comparável entre os três grupos terapêuticos. Pacientes que na linha basal tinham MCV anormal ou normal alto deveriam ter suplementaçao com ácido fólico desde o início