Immunomodulation and fibroblast dynamics driving nociceptive joint pain within inflammatory synovium: Unravelling mechanisms for therapeutic advancements in osteoarthritis.

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. CONCLUSION: 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.

Cross-species transcriptomics identifies obesity associated genes between human and mouse studies.

BACKGROUND: Fundamentally defined by an imbalance in energy consumption and energy expenditure, obesity is a significant risk factor of several musculoskeletal conditions including osteoarthritis (OA). High-fat diets and sedentary lifestyle leads to increased adiposity resulting in systemic inflammation due to the endocrine properties of adipose tissue producing inflammatory cytokines and adipokines. We previously showed serum levels of specific adipokines are associated with biomarkers of bone remodelling and cartilage volume loss in knee OA patients. Whilst more recently we find the metabolic consequence of obesity drives the enrichment of pro-inflammatory fibroblast subsets within joint synovial tissues in obese individuals compared to those of BMI defined 'health weight'. As such this present study identifies obesity-associated genes in OA joint tissues which are conserved across species and conditions. METHODS: The study utilised 6 publicly available bulk and single-cell transcriptomic datasets from human and mice studies downloaded from Gene Expression Omnibus (GEO). Machine learning models were employed to model and statistically test datasets for conserved gene expression profiles. Identified genes were validated in OA tissues from obese and healthy weight individuals using quantitative PCR method (N = 38). Obese and healthy-weight patients were categorised by BMI > 30 and BMI between 18 and 24.9 respectively. Informed consent was obtained from all study participants who were scheduled to undergo elective arthroplasty. RESULTS: Principal component analysis (PCA) was used to investigate the variations between classes of mouse and human data which confirmed variation between obese and healthy populations. Differential gene expression analysis filtered on adjusted p-values of p < 0.05, identified differentially expressed genes (DEGs) in mouse and human datasets. DEGs were analysed further using area under curve (AUC) which identified 12 genes. Pathway enrichment analysis suggests these genes were involved in the biosynthesis and elongation of fatty acids and the transport, oxidation, and catabolic processing of lipids. qPCR validation found the majority of genes showed a tendency to be upregulated in joint tissues from obese participants. Three validated genes, IGFBP2 (p = 0.0363), DOK6 (0.0451) and CASP1 (0.0412) were found to be significantly different in obese joint tissues compared to lean-weight joint tissues. CONCLUSIONS: The present study has employed machine learning models across several published obesity datasets to identify obesity-associated genes which are validated in joint tissues from OA. These results suggest obesity-associated genes are conserved across conditions and may be fundamental in accelerating disease in obese individuals. Whilst further validations and additional conditions remain to be tested in this model, identifying obesity-associated genes in this way may serve as a global aid for patient stratification giving rise to the potential of targeted therapeutic interventions in such patient subpopulations.

Obesity defined molecular endotypes in the synovium of patients with osteoarthritis provides a rationale for therapeutic targeting of fibroblast subsets.

BACKGROUND: Osteoarthritis (OA), a multifaceted condition, poses a significant challenge for the successful clinical development of therapeutics due to heterogeneity. However, classifying molecular endotypes of OA pathogenesis could provide invaluable phenotype-directed routes for stratifying subgroups of patients for targeted therapeutics, leading to greater chances of success in trials. This study establishes endotypes in OA soft joint tissue driven by obesity in both load-bearing and non-load bearing joints. METHODS: Hand, hip, knee and foot joint synovial tissue was obtained from OA patients (n = 32) classified as obese (BMI > 30) or normal weight (BMI 18.5-24.9). Isolated fibroblasts (OA SF) were assayed by Olink proteomic panel, seahorse metabolic flux assay, Illumina's NextSeq 500 bulk and Chromium 10X single cell RNA-sequencing, validated by Luminex and immunofluorescence. RESULTS: Targeted proteomic, metabolic and transcriptomic analysis found the inflammatory landscape of OA SFs are independently impacted by obesity, joint loading and anatomical site with significant heterogeneity between obese and normal weight patients, confirmed by bulk RNAseq. Further investigation by single cell RNAseq identified four functional molecular endotypes including obesity specific subsets defined by an inflammatory endotype related to immune cell regulation, fibroblast activation and inflammatory signaling, with up-regulated CXCL12, CFD and CHI3L1 expression. Luminex confirmed elevated chitase3-like-1(229.5 vs. 49.5 ng/ml, p < .05) and inhibin (20.6 vs. 63.8 pg/ml, p < .05) in obese and normal weight OA SFs, respectively. Lastly, we find SF subsets in obese patients spatially localise in sublining and lining layers of OA synovium and can be distinguished by differential expression of the transcriptional regulators MYC and FOS. CONCLUSION: These findings demonstrate the significance of obesity in changing the inflammatory landscape of synovial fibroblasts in both load bearing and non-load bearing joints. Describing multiple heterogeneous OA SF populations characterised by specific molecular endotypes, which drive heterogeneity in OA disease pathogenesis. These molecular endotypes may provide a route for the stratification of patients in clinical trials, providing a rational for the therapeutic targeting of specific SF subsets in specific patient populations with arthritic conditions.

Oligonucleotide Therapeutics for Age-Related Musculoskeletal Disorders: Successes and Challenges.

Age-related disorders of the musculoskeletal system including sarcopenia, osteoporosis and arthritis represent some of the most common chronic conditions worldwide, for which there remains a great clinical need to develop safer and more efficacious pharmacological treatments. Collectively, these conditions involve multiple tissues, including skeletal muscle, bone, articular cartilage and the synovium within the joint lining. In this review, we discuss the potential for oligonucleotide therapies to combat the unmet clinical need in musculoskeletal disorders by evaluating the successes of oligonucleotides to modify candidate pathological gene targets and cellular processes in relevant tissues and cells of the musculoskeletal system. Further, we discuss the challenges that remain for the clinical development of oligonucleotides therapies for musculoskeletal disorders and evaluate some of the current approaches to overcome these.

The role of extracellular vesicle miRNAs and tRNAs in synovial fibroblast senescence.

Extracellular vesicles are mediators of intercellular communication with critical roles in cellular senescence and ageing. In arthritis, senescence is linked to the activation of a pro-inflammatory phenotype contributing to chronic arthritis pathogenesis. We hypothesised that senescent osteoarthritic synovial fibroblasts induce senescence and a pro-inflammatory phenotype in non-senescent osteoarthritic fibroblasts, mediated through extracellular vesicle cargo. Small RNA-sequencing and mass spectrometry proteomics were performed on extracellular vesicles isolated from the secretome of non-senescent and irradiation-induced senescent synovial fibroblasts. ß-galactosidase staining confirmed senescence in SFs. RNA sequencing identified 17 differentially expressed miRNAs, 11 lncRNAs, 14 tRNAs and one snoRNA and, 21 differentially abundant proteins were identified by mass spectrometry. Bioinformatics analysis of miRNAs identified fibrosis, cell proliferation, autophagy, and cell cycle as significant pathways, tRNA analysis was enriched for signaling pathways including FGF, PI3K/AKT and MAPK, whilst protein analysis identified PAX3-FOXO1, MYC and TFGB1 as enriched upstream regulators involved in senescence and cell cycle arrest. Finally, treatment of non-senescent synovial fibroblasts with senescent extracellular vesicles confirmed the bystander effect, inducing senescence in non-senescent cells potentially through down regulation of NF-κß and cAMP response element signaling pathways thus supporting our hypothesis. Understanding the exact composition of EV-derived small RNAs of senescent cells in this way will inform our understanding of their roles in inflammation, intercellular communication, and as active molecules in the senescence bystander effect.

Differential Metabotypes in Synovial Fibroblasts and Synovial Fluid in Hip Osteoarthritis Patients Support Inflammatory Responses.

Changes in cellular metabolism have been implicated in mediating the activated fibroblast phenotype in a number of chronic inflammatory disorders, including pulmonary fibrosis, renal disease and rheumatoid arthritis. The aim of this study was therefore to characterise the metabolic profile of synovial joint fluid and synovial fibroblasts under both basal and inflammatory conditions in a cohort of obese and normal-weight hip OA patients. Furthermore, we sought to ascertain whether modulation of a metabolic pathway in OA synovial fibroblasts could alter their inflammatory activity. Synovium and synovial fluid was obtained from hip OA patients, who were either of normal-weight or obese and were undergoing elective joint replacement surgery. The synovial fluid metabolome was determined by 1H NMR spectroscopy. The metabolic profile of isolated synovial fibroblasts in vitro was characterised by lactate secretion, oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) using the Seahorse XF Analyser. The effects of a small molecule pharmacological inhibitor and siRNA targeted at glutaminase-1 (GLS1) were assessed to probe the role of glutamine metabolism in OA synovial fibroblast function. Obese OA patient synovial fluid (n = 5) exhibited a different metabotype, compared to normal-weight patient fluid (n = 6), with significantly increased levels of 1, 3-dimethylurate, N-Nitrosodimethylamine, succinate, tyrosine, pyruvate, glucose, glycine and lactate, and enrichment of the glutamine-glutamate metabolic pathway, which correlated with increasing adiposity. In vitro, isolated obese OA fibroblasts exhibited greater basal lactate secretion and aerobic glycolysis, and increased mitochondrial respiration when stimulated with pro-inflammatory cytokine TNFα, compared to fibroblasts from normal-weight patients. Inhibition of GLS1 attenuated the TNFα-induced expression and secretion of IL-6 in OA synovial fibroblasts. These findings suggest that altered cellular metabolism underpins the inflammatory phenotype of OA fibroblasts, and that targeted inhibition of glutamine-glutamate metabolism may provide a route to reducing the pathological effects of joint inflammation in OA patients who are obese.

Long Non-coding RNAs in Rheumatology.

The last decade has seen an enormous increase in long non-coding RNA (lncRNA) research within rheumatology. LncRNAs are arbitrarily classed as non-protein encoding RNA transcripts that exceed 200 nucleotides in length. These transcripts have tissue and cell specific patterns of expression and are implicated in a variety of biological processes. Unsurprisingly, numerous lncRNAs are dysregulated in rheumatoid conditions, correlating with disease activity and cited as potential biomarkers and targets for therapeutic intervention. In this chapter, following an introduction into each condition, we discuss the lncRNAs involved in rheumatoid arthritis, osteoarthritis and systemic lupus erythematosus. These inflammatory joint conditions share several inflammatory signalling pathways and therefore not surprisingly many commonly dysregulated lncRNAs are shared across these conditions. In the interest of translational research only those lncRNAs which are strongly conserved have been addressed. The lncRNAs discussed here have diverse roles in regulating inflammation, proliferation, migration, invasion and apoptosis. Understanding the molecular basis of lncRNA function in rheumatology will be crucial in fully determining the inflammatory mechanisms that drive these conditions.

Synovial tissue from sites of joint pain in knee osteoarthritis patients exhibits a differential phenotype with distinct fibroblast subsets.

BACKGROUND: Synovial inflammation is associated with pain severity in patients with knee osteoarthritis (OA). The aim here was to determine in a population with knee OA, whether synovial tissue from areas associated with pain exhibited different synovial fibroblast subsets, compared to synovial tissue from sites not associated with pain. A further aim was to compare differences between early and end-stage disease synovial fibroblast subsets. METHODS: Patients with early knee OA (n = 29) and end-stage knee OA (n = 22) were recruited. Patient reported pain was recorded by questionnaire and using an anatomical knee pain map. Proton density fat suppressed MRI axial and sagittal sequences were analysed and scored for synovitis. Synovial tissue was obtained from the medial and lateral parapatellar and suprapatellar sites. Fibroblast single cell RNA sequencing was performed using Chromium 10X and analysed using Seurat. Transcriptomes were functionally characterised using Ingenuity Pathway Analysis and the effect of fibroblast secretome on neuronal growth assessed using rat DRGN. FINDINGS: Parapatellar synovitis was significantly associated with the pattern of patient-reported pain in knee OA patients. Synovial tissue from sites of patient-reported pain exhibited a differential transcriptomic phenotype, with distinct synovial fibroblast subsets in early OA and end-stage OA. Functional pathway analysis revealed that synovial tissue and fibroblast subsets from painful sites promoted fibrosis, inflammation and the growth and activity of neurons. The secretome of fibroblasts from early OA painful sites induced greater survival and neurite outgrowth in dissociated adult rodent dorsal root ganglion neurons. INTERPRETATION: Sites of patient-reported pain in knee OA exhibit a different synovial tissue phenotype and distinct synovial fibroblast subsets. Further interrogation of these fibroblast pathotypes will increase our understanding of the role of synovitis in OA joint pain and provide a rationale for the therapeutic targeting of fibroblast subsets to alleviate pain in patients. FUNDING: This study was funded by Versus Arthritis, UK (21530; 21812).

Oligonucleotide Therapies in the Treatment of Arthritis: A Narrative Review.

Osteoarthritis (OA) and rheumatoid arthritis (RA) are two of the most common chronic inflammatory joint diseases, for which there remains a great clinical need to develop safer and more efficacious pharmacological treatments. The pathology of both OA and RA involves multiple tissues within the joint, including the synovial joint lining and the bone, as well as the articular cartilage in OA. In this review, we discuss the potential for the development of oligonucleotide therapies for these disorders by examining the evidence that oligonucleotides can modulate the key cellular pathways that drive the pathology of the inflammatory diseased joint pathology, as well as evidence in preclinical in vivo models that oligonucleotides can modify disease progression.

The Expression and Function of Metastases Associated Lung Adenocarcinoma Transcript-1 Long Non-Coding RNA in Subchondral Bone and Osteoblasts from Patients with Osteoarthritis.

Metastasis Associated Lung Adenocarcinoma Transcript-1 (MALAT1) is implicated in regulating the inflammatory response and in the pathology of several chronic inflammatory diseases, including osteoarthritis (OA). The purpose of this study was to examine the relationship between OA subchondral bone expression of MALAT1 with parameters of joint health and biomarkers of joint inflammation, and to determine its functional role in human OA osteoblasts. Subchondral bone and blood were collected from hip and knee OA patients (n = 17) and bone only from neck of femur fracture patients (n = 6) undergoing joint replacement surgery. Cytokines were determined by multiplex assays and ELISA, and gene expression by qPCR. MALAT1 loss of function was performed in OA patient osteoblasts using locked nucleic acids. The osteoblast transcriptome was analysed by RNASeq and pathway analysis. Bone expression of MALAT1 positively correlated to serum DKK1 and galectin-1 concentrations, and in OA patient osteoblasts was induced in response to IL-1ß stimulation. Osteoblasts depleted of MALAT1 exhibited differential expression (>1.5 fold change) of 155 genes, including PTGS2. Both basal and IL-1ß-mediated PGE2 secretion was greater in MALAT1 depleted osteoblasts. The induction of MALAT1 in human OA osteoblasts upon inflammatory challenge and its modulation of PGE2 production suggests that MALAT1 may play a role in regulating inflammation in OA subchondral bone.

A long intergenic non-coding RNA regulates nuclear localization of DNA methyl transferase-1.

DNA methyl transferase-1 or DNMT1 maintains DNA methylation in the genome and is important for regulating gene expression in cells. Aberrant changes in DNMT1 activity and DNA methylation are commonly observed in cancers and many other diseases. Recently, a number of long intergenic non-protein-coding RNAs or lincRNAs have been shown to play a role in regulating DNMT1 activity. CCDC26 is a nuclear lincRNA that is frequently mutated in cancers and is a hotbed for disease-associated single nucleotide changes. However, the functional mechanism of CCDC26 is not understood. Here, we show that this lincRNA is concentrated on the nuclear periphery. Strikingly, in the absence of CCDC26 lincRNA, DNMT1 is mis-located in the cytoplasm, and the genomic DNA is significantly hypomethylated. This is accompanied by double-stranded DNA breaks and increased cell death. These results point to a previously unrecognized mechanism of lincRNA-mediated subcellular localization of DNMT1 and regulation of DNA methylation.

Novel gene variants in patients with platelet-based bleeding using combined exome sequencing and RNAseq murine expression data.

Essentials Identifying genetic variants in platelet disorders is challenging due to its heterogenous nature. We combine WES, RNAseq, and python-based bioinformatics to identify novel gene variants. We find novel candidates in patient data by cross-referencing against a murine RNAseq model of thrombopoiesis. This innovative combined bioinformatic approach provides novel data for future research in the field. ABSTRACT: Background The UK Genotyping and Phenotyping of Platelets study has recruited and analyzed 129 patients with suspected heritable bleeding. Previously, 55 individuals had a definitive genetic diagnosis based on whole exome sequencing (WES) and platelet morphological and functional testing. A significant challenge in this field is defining filtering criteria to identify the most likely candidate mutations for diagnosis and further study. Objective Identify candidate gene mutations for the remaining 74 patients with platelet-based bleeding with unknown genetic cause, forming the basis of future re-recruitment and further functional testing and assessment. Methods Using python-based data frame indexing, we first identify and filter all novel and rare variants using a panel of 116 genes known to cause bleeding across the full cohort of WES data. This identified new variants not previously reported in this cohort. We then index the remaining patients, with rare or novel variants in known bleeding genes against a murine RNA sequencing dataset that models proplatelet-forming megakaryocytes. Results Filtering against known genes identified candidate variants in 59 individuals, including novel variants in several known genes. In the remaining cohort of "unknown" patients, indexing against differentially expressed genes revealed candidate gene variants in several novel unreported genes, focusing on 14 patients with a severe clinical presentation. Conclusions We identified candidate mutations in a cohort of patients with no previous genetic diagnosis. This work involves innovative coupling of RNA sequencing and WES to identify candidate variants forming the basis of future study in a significant number of undiagnosed patients.

Involvements of long noncoding RNAs in obesity-associated inflammatory diseases.

Obesity is associated with chronic low-grade inflammation that affects the phenotype of multiple tissues and therefore is implicated in the development and progression of several age-related chronic inflammatory disorders. Importantly, a new family of noncoding RNAs, termed long noncoding RNAs (lncRNAs), have been identified as key regulators of inflammatory signalling pathways that can mediate both pretranscriptional and posttranscriptional gene regulation. Furthermore, several lncRNAs have been identified, which are differentially expressed in multiple tissue types in individuals who are obese or in preclinical models of obesity. In this review, we examine the evidence for the role of several of the most well-studied lncRNAs in the regulation of inflammatory pathways associated with obesity. We highlight the evidence for their differential expression in the obese state and in age-related conditions including insulin resistance, type 2 diabetes (T2D), sarcopenia, osteoarthritis and rheumatoid arthritis, where obesity plays a significant role. Determining the expression and functional role of lncRNAs in mediating obesity-associated chronic inflammation will advance our understanding of the epigenetic regulatory pathways that underlie age-related inflammatory diseases and may also ultimately identify new targets for therapeutic intervention.

Regulation of the Inflammatory Synovial Fibroblast Phenotype by Metastasis-Associated Lung Adenocarcinoma Transcript 1 Long Noncoding RNA in Obese Patients With Osteoarthritis.

OBJECTIVE: To identify long noncoding RNAs (lncRNAs) associated with the inflammatory phenotype of synovial fibroblasts from obese patients with osteoarthritis (OA), and to explore the expression and function of these lncRNAs. METHODS: Synovium was collected from normal-weight patients with hip fracture (non-OA; n = 6) and from normal-weight (n = 8) and obese (n = 8) patients with hip OA. Expression of RNA was determined by RNA-sequencing and quantitative reverse transcription-polymerase chain reaction. Knockdown of lncRNA was performed using LNA-based GapmeRs. Synovial fibroblast cytokine production was measured by enzyme-linked immunosorbent assay. RESULTS: Synovial fibroblasts from obese patients with OA secreted greater levels of interleukin-6 (IL-6) (mean ± SEM 162 ± 21 pg/ml; P < 0.001) and CXCL8 (262 ± 67 pg/ml; P < 0.05) compared to fibroblasts from normal-weight patients with OA (IL-6, 51 ± 4 pg/ml; CXCL8, 78 ± 11 pg/ml) or non-OA patients (IL-6, 35 ± 3 pg/ml; CXCL8, 56 ± 6 pg/ml) (n = 6 patients per group). RNA-sequencing revealed that fibroblasts from obese OA patients exhibited an inflammatory transcriptome, with increased expression of proinflammatory messenger RNAs (mRNAs) as compared to that in fibroblasts from normal-weight OA or non-OA patients (>2-fold change, P < 0.05; n = 4 patients per group). A total of 19 lncRNAs were differentially expressed between normal-weight OA and non-OA patient fibroblasts, and a further 19 lncRNAs were differentially expressed in fibroblasts from obese OA patients compared to normal-weight OA patients (>2-fold change, P < 0.05 for each), which included the lncRNA for metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). MALAT1 was rapidly induced upon stimulation of OA synovial fibroblasts with proinflammatory cytokines, and was up-regulated in the synovium from obese OA patients as compared to normal-weight OA patients (1.6-fold change, P < 0.001) or non-OA patients (6-fold change, P < 0.001). MALAT1 knockdown in OA synovial fibroblasts (n = 4 patients) decreased the levels of mRNA expression and protein secretion of CXCL8 (>1.5-fold change, P < 0.01), whereas it increased expression of mRNAs for TRIM6 (>2-fold change, P < 0.01), IL7R (<2-fold change, P < 0.01), HIST1H1C (>1.5-fold change, P < 0.001), and MAML3 (>1.5-fold change, P < 0.001). In addition, MALAT1 knockdown inhibited the proliferation of synovial fibroblasts from obese patients with OA. CONCLUSION: Synovial fibroblasts from obese patients with hip OA exhibit an inflammatory phenotype. MALAT1 lncRNA may mediate joint inflammation in obese OA patients.

The combination of local infiltration analgesia reagents increases their detrimental effect on human hip OA patient osteoblast viability and function.

The purpose was to evaluate the effect of Local infiltration analgesia (LIA) reagents in monotherapy and in combinations at clinical doses, on the viability and function of osteoblasts isolated from hip OA patients undergoing orthopaedic surgery. Human hip OA osteoblasts were exposed to LIA reagents including Bupivacaine, Lidocaine, Ropivacaine, Ketorolac and combinations with Adrenaline for 30 min. Osteoblast cellular viability and function was determined at 24 h and 7 days post-exposure. In conclusion, our data shows that LIA reagents, most notably Bupivacaine and its use in combination, are detrimental to human hip OA osteoblasts at concentrations advocated for clinical use.

A novel form of JARID2 is required for differentiation in lineage-committed cells.

Polycomb repressive complex-2 (PRC2) is a group of proteins that play an important role during development and in cell differentiation. PRC2 is a histone-modifying complex that catalyses methylation of lysine 27 of histone H3 (H3K27me3) at differentiation genes leading to their transcriptional repression. JARID2 is a co-factor of PRC2 and is important for targeting PRC2 to chromatin. Here, we show that, unlike in embryonic stem cells, in lineage-committed human cells, including human epidermal keratinocytes, JARID2 predominantly exists as a novel low molecular weight form, which lacks the N-terminal PRC2-interacting domain (ΔN-JARID2). We show that ΔN-JARID2 is a cleaved product of full-length JARID2 spanning the C-terminal conserved jumonji domains. JARID2 knockout in keratinocytes results in up-regulation of cell cycle genes and repression of many epidermal differentiation genes. Surprisingly, repression of epidermal differentiation genes in JARID2-null keratinocytes can be rescued by expression of ΔN-JARID2 suggesting that, in contrast to PRC2, ΔN-JARID2 promotes activation of differentiation genes. We propose that a switch from expression of full-length JARID2 to ΔN-JARID2 is important for the up-regulation differentiation genes.