Fibroblast-like synoviocytes from fluid and synovial membrane from primary osteoarthritis demonstrate similar production of interleukin 6, and metalloproteinases 1 and 3.

OBJECTIVES: The objective of the study was to compare the production of metalloproteinases (MMP)-1, -3 and interleukin (IL)-6 by fibroblast-like synoviocytes (FLS) derived from synovial fluid (FD-FLS), and FLS derived from synovial tissue (TD-FLS) of patients with primary osteoarthritis (OA). The more accessible FD-FLS could facilitate the study of the role of these cells in OA pathophysiology. METHODS: MMP-1, MMP-3, and IL-6 levels were measured in the supernatant culture at baseline and 22 hours after stimulation with TNF-α and IL-1 ß. RESULTS: There was no difference at baseline between MMP-1, MMP-3 and IL-6 production by FD-FLS and TDFLS. Analogous to baseline, stimulation of FD-FLS and TD-FLS with IL-1ß and TNF-α did not result in difference on MMP-3 and IL-6 production. However, TD-FLS produced more MMP-1 than FD-FLS after stimulation with IL-1ß (p=0.01). Additionally, there was a positive correlation for production of MMP-1, MMP-3 and IL-6 between FD-FLS and TD-FLS (r=0.40 and p<0.0008; r=0.66 and p<0.0001; r=0.76 and p<0.0001, respectively). Supporting this statistical significant positive correlation, the Bland-Altman plotting, showed a homogeneous distribution of the values and low mean disagreement rates between all results of FD-FLS and TD-FLS (23.1%, 56.8% and 48.1%, respectively). CONCLUSIONS: Our data demonstrated functional similarity between FD-FLS and TD-FLS and support the use of a more accessible source of FLS for the study of the pathogenesis of joint destruction and therapeutic targets in primary OA.

Joint-specific rheumatoid arthritis fibroblast-like synoviocyte regulation identified by integration of chromatin access and transcriptional activity.

The mechanisms responsible for the distribution and severity of joint involvement in rheumatoid arthritis (RA) are not known. To explore whether site-specific FLS biology might be associated with location-specific synovitis and explain the predilection for hand (wrist/metacarpal phalangeal joints) involvement in RA, we generated transcriptomic and chromatin accessibility data from FLS to identify the transcription factors (TFs) and pathways. Networks were constructed by integration of chromatin accessibility and gene expression data. Analysis revealed joint-specific patterns of FLS phenotype, with proliferative, migratory, proinflammatory, and matrix-degrading characteristics observed in resting FLS derived from the hand joints compared with hip or knee. TNF-stimulation amplified these differences, with greater enrichment of proinflammatory and proliferative genes in hand FLS compared with hip and knee FLS. Hand FLS also had the greatest expression of markers associated with an 'activated' state relative to the 'resting' state, with the greatest cytokine and MMP expression in TNF-stimulated hand FLS. Predicted differences in proliferation and migration were biologically validated with hand FLS exhibiting greater migration and cell growth than hip or knee FLS. Distinctive joint-specific FLS biology associated with a more aggressive inflammatory response might contribute to the distribution and severity of joint involvement in RA.

Dysregulated NUB1 and Neddylation Enhances Rheumatoid Arthritis Fibroblast-Like Synoviocyte Inflammatory Responses.

OBJECTIVE: Fibroblast-like synoviocytes (FLS) contribute to the pathogenesis of rheumatoid arthritis (RA), in part due to activation of the proinflammatory transcription factor NF-κB. Neddylation is modulated by the negative regulator of ubiquitin-like protein (NUB) 1. We determined whether NUB1 and neddylation are aberrant in the models with RA FLS, thereby contributing to their aggressive phenotype. METHODS: Models with RA or osteoarthritis (OA) FLS were obtained from arthroplasty synovia. Real-time quantitative polymerase chain reaction and Western blot analysis assessed gene and protein expression, respectively. NUB1 was overexpressed using an expression vector. NF-κB activation was assessed by stimulating FLS with interleukin (IL)-1ß. Neddylation inhibitor (MLN4924) and proteasome inhibitor were used in migration and gene expression assays. MLN4924 was used in the model with K/BxN serum-transfer arthritis. RESULTS: Enhanced H3K27ac and H3K27me3 peaks were observed in the NUB1 promoter in the OA FLS compared with the RA FLS. NUB1 was constitutively expressed by FLS, but induction by IL-1ß was significantly greater in the OA FLS. The ratio of neddylated cullin (CUL) 1 to nonneddylated CUL1 was lower in the OA FLS than the RA FLS. NUB1 overexpression decreased NF-κB nuclear translocation and IL-6 messenger RNA (mRNA) in IL-1ß-stimulated the RA FLS. MLN4924 decreased CUL1 neddylation, NF-κB nuclear translocation, and IL-6 mRNA in IL-1ß-stimulated the RA FLS. MLN4924 significantly decreased arthritis severity in the model with K/BxN serum-transfer arthritis. CONCLUSION: CUL1 neddylation and NUB1 induction is dysregulated in the models with RA, which increases FLS activation. Inhibition of neddylation is an effective therapy in an animal model of arthritis. These data suggest that the neddylation system contributes to the pathogenesis of RA and that regulation of neddylation could be a novel therapeutic approach.

Joint-specific regulation of homeobox D10 expression in rheumatoid arthritis fibroblast-like synoviocytes.

Rheumatoid arthritis (RA) is a systemic immune-mediated disease characterized by joint inflammation and destruction. The disease typically affects small joints in the hands and feet, later progressing to involve larger joints such as the knees, shoulders, and hips. While the reasons for these joint-specific differences are unclear, distinct epigenetic patterns associated with joint location have been reported. In this study, we evaluated the unique epigenetic landscapes of fibroblast-like synoviocytes (FLS) from hip and knee synovium in RA patients, focusing on the expression and regulation of Homeobox (HOX) transcription factors. These highly conserved genes play a critical role in embryonic development and are known to maintain distinct expression patterns in various adult tissues. We found that several HOX genes, especially HOXD10, were differentially expressed in knee FLS compared with hip FLS. Epigenetic differences in chromatin accessibility and histone marks were observed in HOXD10 promoter between knee and hip FLS. Histone modification, particularly histone acetylation, was identified as an important regulator of HOXD10 expression. To understand the mechanism of differential HOXD10 expression, we inhibited histone deacetylases (HDACs) with small molecules and siRNA. We found that HDAC1 blockade or deficiency normalized the joint-specific HOXD10 expression patterns. These observations suggest that epigenetic differences, specifically histone acetylation related to increased HDAC1 expression, play a crucial role in joint-specific HOXD10 expression. Understanding these mechanisms could provide insights into the regional aspects of RA and potentially lead to therapeutic strategies targeting specific patterns of joint involvement during the course of disease.

Laser Capture Microscopy RNA Sequencing for Topological Mapping of Synovial Pathology During Rheumatoid Arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) is an autoimmune disease in which the joint lining or synovium becomes highly inflamed and majorly contributes to disease progression. Understanding pathogenic processes in RA synovium is critical for identifying therapeutic targets. We performed laser capture microscopy (LCM) followed by RNA sequencing (LCM-RNAseq) to study regional transcriptomes throughout RA synovium. METHODS: Synovial lining, sublining, and vessel samples were captured by LCM from seven patients with RA and seven patients with osteoarthritis (OA). RNAseq was performed on RNA extracted from captured tissue. Principal component analysis was performed on the sample set by disease state. Differential expression analysis was performed between disease states based on log2 fold change and q value parameters. Pathway analysis was performed using the Reactome Pathway Database on differentially expressed genes among disease states. Significantly enriched pathways in each synovial region were selected based on the false discovery rate. RESULTS: RA and OA transcriptomes were distinguishable by principal component analysis. Pairwise comparisons of synovial lining, sublining, and vessel samples between RA and OA revealed substantial differences in transcriptional patterns throughout the synovium. Hierarchical clustering of pathways based on significance revealed a pattern of association between biologic function and synovial topology. Analysis of pathways uniquely enriched in each region revealed distinct phenotypic abnormalities. As examples, RA lining samples were marked by anomalous immune cell signaling, RA sublining samples were marked by aberrant cell cycle, and RA vessel samples were marked by alterations in heme scavenging. CONCLUSION: LCM-RNAseq confirms reported transcriptional differences between the RA synovium and the OA synovium and provides evidence supporting a relationship between synovial topology and molecular anomalies in RA.