miRNAs constitute fine-tuners of gene expression and are implicated in a variety of diseases spanning from inflammation to cancer. miRNA expression is deregulated in rheumatoid arthritis (RA); however, their specific role in key arthritogenic cells such as the synovial fibroblast (SF) remains elusive. Previous studies have shown that Mir221/222 expression is upregulated in RA SFs. Here, we demonstrate that TNF and IL-1β but not IFN-γ activated Mir221/222 gene expression in murine SFs. SF-specific overexpression of Mir221/222 in huTNFtg mice led to further expansion of SFs and disease exacerbation, while its total ablation led to reduced SF expansion and attenuated disease. Mir221/222 overexpression altered the SF transcriptional profile igniting pathways involved in cell cycle and ECM (extracellular matrix) regulation. Validation of targets of Mir221/222 revealed cell cycle inhibitors Cdkn1b and Cdkn1c, as well as the epigenetic regulator Smarca1. Single-cell ATAC-seq data analysis revealed increased Mir221/222 gene activity in pathogenic SF subclusters and transcriptional regulation by Rela, Relb, Junb, Bach1, and Nfe2l2. Our results establish an SF-specific pathogenic role of Mir221/222 in arthritis and suggest that its therapeutic targeting in specific subpopulations could lead to novel fibroblast-targeted therapies.

Temporomandibular joint osteoarthritis (TMJOA) is considered to be a low-grade inflammatory disease involving multiple joint tissues. The crosstalk between synovium and cartilage plays an important role in TMJOA. Synovial cells are a group of heterogeneous cells and synovial microenvironment is mainly composed of synovial fibroblasts (SF) and synovial macrophages. In TMJOA, SF and synovial macrophages release a large number of inflammatory cytokines and extracellular vesicles and promote cartilage destruction. Cartilage wear particles stimulate SF proliferation and macrophages activation and exacerbate synovitis. In TMJOA, chondrocytes and synovial cells exhibit increased glycolytic activity and lactate secretion, leading to impaired chondrocyte matrix synthesis. Additionally, the synovium contains mesenchymal stem cells, which are the seed cells for cartilage repair in TMJOA. Co-culture of chondrocytes and synovial mesenchymal stem cells enhances the chondrogenic differentiation of stem cells. This review discusses the pathological changes of synovium in TMJOA, the means of crosstalk between synovium and cartilage, and their influence on each other. Based on the crosstalk between synovium and cartilage in TMJOA, we illustrate the treatment strategies for improving synovial microenvironment, including reducing cell adhesion, utilizing extracellular vesicles to deliver biomolecules, regulating cellular metabolism and targeting inflammatory cytokines.

Rheumatoid arthritis (RA) is a common autoimmune condition and chronic inflammatory disease, mostly affecting synovial joints. The complex pathogenesis of RA is supportive of high morbidity, disability, and mortality rates. Pathological changes a common characteristic in RA synovial tissue is attributed to the inadequacy of apoptotic pathways. In that regard, apoptotic pathways have been the center of attention in RA therapeutic approaches. As the regulators in the complex network of apoptosis, microRNAs (miRNAs) are found to be vital modulators in both intrinsic and extrinsic pathways through altering their regulatory genes. Indeed, miRNA, a member of the family of non-coding RNAs, are found to be an important player in not even apoptosis, but proliferation, gene expression, signaling pathways, and angiogenesis. Aberrant expression of miRNAs is implicated in attenuation and/or intensification of various apoptosis routes, resulting in culmination of human diseases including RA. Considering the need for more studies focused on the underlying mechanisms of RA in order to elevate the unsatisfactory clinical treatments, this study is aimed to delineate the importance of apoptosis in the pathophysiology of this disease. As well, this review is focused on the critical role of miRNAs in inducing or inhibiting apoptosis of RA-synovial fibroblasts and fibroblast-like synoviocytes and how this mechanism can be exerted for therapeutic purposes for RA.

BACKGROUND: Hypocortisolemia is associated with increased expression of NR3C1 (glucocorticoid receptor, GR) in blood cells. As endogenous cortisol production is decreased in some RA patients, we tested the hypothesis that GR may be aberrantly expressed in rheumatoid synovium.
METHODS: We defined the cellular pattern of NR3C1 synovial expression using human and mouse single-cell RNA-sequencing data. Bulk synovial RNA-sequencing data from early (n = 57) or established (n = 94) RA were compared to osteoarthritis (n = 22) and healthy synovium (n = 28).
RESULTS: GR was expressed in all synovial cell types in both human and experimental arthritis. GR synovial expression, as well as 11β-HSD1/11β-HSD2 enzyme ratio, were higher in RA than healthy and osteoarthritic tissue, regardless of disease duration or treatment. Given that GR expression varied across samples, we searched for differences between RA patients with higher versus lower GR expression. Indeed, the synovial transcriptome of RA patients with high versus low GR expression (1st quartile, 30,517 ± 4876 vs. 4th quartile, 19,382 ± 2523 normalized counts) was enriched for proinflammatory gene-sets, including \'inflammatory response\', \'IFN-γ response\' and \'IL6/JAK/STAT3 signalling\'. High synovial GR expression was also associated with increased JAK2 and PTPRK expression, denoting activation of the proinflammatory sublining fibroblasts. In contrast, low GR expression was associated with increased COMP and COL6A2 expression, denoting a resting synovial state.
CONCLUSIONS: GR is overexpressed in the synovium of some RA patients in association with proinflammatory gene expression and activated sublining fibroblast status. Further studies should examine whether GR overexpression may act as a compensatory mechanism sensitizing synovial tissue to glucocorticoid action in RA.

Pulsed electromagnetic field stimulation (PEMF) is gaining more attention as a non-invasive arthritis treatment. In our study, immortalized synovial fibroblasts (K4IM) derived from a non-arthritic donor were exposed to MAGCELL® ARTHRO, a PEMF device, with 105 mT intensity, 8 Hz frequency, and 2 × 2.5 min sessions conducted thrice with a 1 h interval, to understand the underlying mechanism in regard to the complement system. Additionally, tumor necrosis factor (TNFα, 10 ng/mL) pre-treatment prior to PEMF stimulation, as well as 3-day versus 6-day stimulation, were compared. Gene expression of C4b binding protein-alpha and -beta (C4BPα, C4BPβ), complement factor (CF)-H, CFI, CD55, CD59, Interleukin (IL-6) and TNFα was analyzed. Immunofluorescence staining of CD55, CD59, and Ki67 was conducted. Results showed the absence of C4BPα gene expression, but C4BPβ was present. One and three days of PEMF stimulation caused no significant changes. However, after six days, there was a significant increase in CD55, CFH, and CD59 gene expression, indicating cytoprotective effects. Conversely, IL-6 gene expression increased after six days of stimulation and even after a single session in TNFα pre-stimulated cells, indicating a pro-inflammatory effect. PEMF\'s ambivalent, i.e., enhancing complement regulatory proteins and pro-inflammatory cytokines, highlights its complexity at the molecular level.

OBJECTIVE: Synovitis is a widely accepted sign of osteoarthritis (OA), characterised by tissue hyperplasia, where increased infiltration of immune cells and proliferation of resident fibroblasts adopt a pro-inflammatory phenotype, and increased the production of pro-inflammatory mediators that are capable of sensitising and activating sensory nociceptors, which innervate the joint tissues. As such, it is important to understand the cellular composition of synovium and their involvement in pain sensitisation to better inform the development of effective analgesics.
METHODS: Studies investigating pain sensitisation in OA with a focus on immune cells and fibroblasts were identified using PubMed, Web of Science and SCOPUS.
RESULTS: In this review, we comprehensively assess the evidence that cellular crosstalk between resident immune cells or synovial fibroblasts with joint nociceptors in inflamed OA synovium contributes to peripheral pain sensitisation. Moreover, we explore whether the elucidation of common mechanisms identified in similar joint conditions may inform the development of more effective analgesics specifically targeting OA joint pain.
CONCLUSIONS: The concept of local environment and cellular crosstalk within the inflammatory synovium as a driver of nociceptive joint pain presents a compelling opportunity for future research and therapeutic advancements.

Calcium pyrophosphate dehydrate (CPPD) crystals are found in the synovial fluid of patients with articular chondrocalcinosis or sometimes with osteoarthritis. In inflammatory conditions, the synovial membrane (SM) is subjected to transient hypoxia, especially during movement. CPPD formation is supported by an increase in extracellular inorganic pyrophosphate (ePPi) levels, which are mainly controlled by the transporter Ank and ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1). We demonstrated previously that transforming growth factor (TGF)-β1 increased ePPi production by inducing Ank and Enpp1 expression in chondrocytes. As the TGF-β1 level raises in synovial fluid under hypoxic conditions, we investigated whether hypoxia may transform SM as a major source of ePPi production. Synovial fibroblasts and SM explants were exposed to 10 ng/mL of TGF-β1 in normoxic or hypoxic (5% O2) culture conditions. Ank and Enpp1 expression were assessed by quantitative PCR, Western blot and immunohistochemistry. ePPi was quantified in culture supernatants. RNA silencing was used to define the respective roles of Ank and Enpp1 in TGF-β1-induced ePPi generation. The molecular mechanisms involved in hypoxia were investigated using an Ank promoter reporter plasmid for transactivation studies, as well as gene overexpression and RNA silencing, the respective role of hypoxia-induced factor (HIF)-1 and HIF-2. Our results showed that TGF-β1 increased Ank, Enpp1, and therefore ePPi production in synovial fibroblasts and SM explants. Ank was the major contributor in ePPi production compared to ENPP1. Hypoxia increased ePPi levels on its own and enhanced the stimulating effect of TGF-β1. Hypoxic conditions enhanced Ank promoter transactivation in an HIF-1-dependent/HIF-2-independent fashion. We demonstrated that under hypoxia, SM is an important contributor to ePPi production in the joint through the induction of Enpp1 and Ank. These findings are of interest as a rationale for the beneficial effect of anti-inflammatory drugs on SM in crystal depositions.

Rheumatoid arthritis (RA) is an autoimmune disease with complex etiology, and its pathological mechanism remains unclear. Our aim was to explore the effect of protein succinylation on RA by silencing Sirt5, sequencing succinylated proteins, and analyzing the sequencing results to identify potential biomarkers. We wanted to gain a clearer understanding of RA pathogenesis, quantitative assessment of succinylated proteins in Fibroblast-like synoviocytes (FLS) from RA patients using liquid chromatography- tandem mass spectrometry and enrichment analysis investigated using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). A total of 679 proteins and 2,471 lysine succinylation sites were found in RA patients, and 436 differentially expressed proteins and 1,548 differentially expressed succinylation sites were identified. Among them, 48 succinylation sites were upregulated in 38 proteins and 144 succinylation sites were downregulated in 82 proteins. Bioinformatics showed that succinylated proteins were significantly enriched in amino and fatty acid metabolisms. Results indicated that Sirt5 can affect various biological processes involved in RA FLSs, and succinylation caused by silencing Sirt5 plays a major role in RA progression. This study provides further understanding of RA pathogenesis and may facilitate searching for potential RA biomarkers.

BACKGROUND: Current strategies that target cytokines (e.g., tumor necrosis factor (TNF)-α), or signaling molecules (e.g., Janus kinase (JAK)) have advanced the management for allergies and autoimmune diseases. Nevertheless, the molecular mechanism that underpins its clinical efficacy have largely remained elusive, especially in the local tissue environment. Here, we aimed to identify the genetic, epigenetic, and immunological targets of JAK inhibitors (JAKis), focusing on their effects on synovial fibroblasts (SFs), the major local effectors associated with destructive joint inflammation in rheumatoid arthritis (RA).
METHODS: SFs were activated by cytokines related to inflammation in RA, and were treated with three types of JAKis or a TNF-α inhibitor (TNFi). Dynamic changes in transcriptome and chromatin accessibility were profiled across samples to identify drug targets. Furthermore, the putative targets were validated using luciferase assays and clustered regularly interspaced short palindromic repeat (CRISPR)-based genome editing.
RESULTS: We found that both JAKis and the TNFi targeted the inflammatory module including IL6. Conversely, specific gene signatures that were preferentially inhibited by either of the drug classes were identified. Strikingly, RA risk enhancers for CD40 and TRAF1 were distinctively regulated by JAKis and the TNFi. We performed luciferase assays and CRISPR-based genome editing, and successfully fine-mapped the single causal variants in these loci, rs6074022-CD40 and rs7021049-TRAF1.
CONCLUSIONS: JAKis and the TNFi had a direct impact on different RA risk enhancers, and we identified nucleotide-resolution targets for both drugs. Distinctive targets of clinically effective drugs could be useful for tailoring the application of these drugs and future design of more efficient treatment strategies.

Rheumatoid arthritis (RA) is a progressive autoimmune disease accompanied by joint swelling, cartilage erosion and bone damage. Drug therapy for RA has been restricted due to poor therapeutic effect, recurrence and adverse effects. Macrophages and synovial fibroblasts both play important roles in the pathology of RA. Macrophages secrete large amount of pro-inflammatory cytokines, while synovial fibroblasts are tightly correlated with hypoxia synovium microenvironment, cytokine release, recruitment of pro-inflammatory cells, bone and cartilage erosion. Therefore, in this timely research, an injectable and pH-sensitive peptide hydrogel loading methotrexate (MTX) and bismuthene nanosheet/polyethyleneimine (BiNS/PEI) has been developed to reduce the activity of macrophages and eliminate over-proliferated synovial fibroblasts simultaneously. MTX can reduce the cytokine secretion of macrophages/anti-apoptosis property of synovial fibroblasts and BiNS/PEI can eliminate synovial fibroblasts via photodynamic therapy (PDT) and photothermal therapy (PTT) routes. The hydrogel was injected into the acidic inflammatory synovium for precise targeting and served as a drug reservoir for pH responsive and sustained drug release, while improving the bioavailability and reducing the toxicity of MTX. Excellent therapeutic efficacy has been achieved in both in vivo and in vitro studies, and this unique drug delivery system provides a new and robust strategy to eliminate synovial fibroblasts and modulate immune system for RA treatment in clinical.