Obese osteoarthritis patients exhibit an inflammatory synovial fibroblast phenotype, which is regulated by the long non coding RNA MALAT1

Objective. To identify lncRNAs associated with the inflammatory phenotype of obese OA synovial fibroblasts and explore their expression and function. Methods. Synovium was collected from normal-weight hip fracture non-OA patients (n=6) and from normal-weight (n=8) and obese hip OA patients (n=8). RNA expression was determined by RNA sequencing and qRT-PCR. LncRNA knockdown utilised LNA-based GapmeRs. Cytokine production was measured by ELISA. Results. Synovial fibroblasts (n=6 patients) from obese OA patients secreted greater IL6 (162 ± 21 pg/ml; p<0.001) and CXCL8 (262 ± 67 pg/ml; p<0.05) than fibroblasts from normal-weight OA (51 ± 4 pg/ml IL6; 78 ± 11 pg/ml CXCL8) or from non-OA patients (35 ± 3 pg/ml IL6; 56 ± 6 pg/ml CXCL8). Sequencing (n=4 patients) revealed obese OA fibroblasts to exhibit an inflammatory transcriptome, with increased expression of proinflammatory mRNAs, compared to normal-weight OA or non-OA fibroblasts. 19 lncRNAs were differentially expressed between normal-weight OA and non-OA fibroblasts and a further 19 lncRNAs differentially expressed between obese OA and normal-weight OA fibroblasts (>2-fold, p<0.05), which included MALAT1. MALAT1 was rapidly induced upon pro-inflammatory cytokine stimulation and upregulated in obese OA synovium, compared to normal-weight OA (1.6-fold, p<0.001) or non-OA synovium (6-fold, p<0.001). MALAT1 knockdown in OA synovial fibroblasts (n=4 patients) decreased CXCL8 expression and secretion (>1.5-fold, p<0.01), increased TRIM6 (>2-fold, p<0.01), IL7R (<2-fold, p<0.01), HIST1H1C (>1.5-fold, p<0.001) and MAML3 (>1.5-fold, p<0.001) expression, and inhibited synovial fibroblast proliferation. Conclusion. Synovial fibroblasts from obese OA patients exhibit an inflammatory phenotype. MALAT1 lncRNA may mediate joint inflammation in obese OA patients. A cc ep te d A rt ic le © 2019 The Authors. Arthritis & Rheumatology published by Wiley Periodicals, Inc. on behalf of American College of Rheumatology


Introduction
OA has historically been considered a wear-and-tear disease of the articular cartilage. In contrast to rheumatoid arthritis (RA), where synovial inflammation (synovitis) is an active driver of disease (1) and where targeting synovial fluid pro-inflammatory cytokines is the rationale behind many of the existing RA therapeutics, OA is often referred to as being a non-inflammatory joint disease. As such, OA drug development has predominantly focused on directly targeting the catabolic and anabolic pathways of cartilage tissue, which has been of limited success (2). However, there is now increasing evidence that synovitis plays a significant role in OA joint pathology (2)(3)(4) by exacerbating cartilage damage via the induction of matrixmetalloproteases (MMPs) and aggrecanases (5)(6)(7), and hastening the onset of end-stage disease. MRI and histological analysis show that synovitis is present at all stages of OA pathogenesis (8)(9)(10), with hyperplasia of the synovial lining (11,12), infiltration of immune cells (13,14) and expression of proinflammatory cytokines (15)(16)(17). The presence of synovitis in early OA, in patients with minimal radiographic signs of cartilage loss (11), suggests that synovitis may represent an opportune point for early therapeutic intervention (3).
We recently reported that the synovial fluid in OA patients who are obese contains greater levels of IL-6, TNF-α and CXCL8, compared to the synovial fluid from normalweight OA patients, and that isolated synovial fibroblasts from obese OA patients secrete more IL-6 (15). Obesity may therefore drive synovial fibroblasts to adopt a more inflammatory phenotype, contributing to an inflammatory joint environment to which the cartilage is exposed. As such, synovitis may play a particularly significant role in the onset and progression of OA in obese individuals, with implications for patient stratification in clinical testing of anti-inflammatory therapeutics (3). Furthermore, determining how obesity-associated inflammation within synovial joint tissues is regulated may lead to the development of new anti-inflammatory therapeutics, which could be of benefit to patients diagnosed with OA or prevent the onset of disease in "at-risk" obese patient populations.
In attempting to better understand the cellular regulators of synovial joint inflammation, long non-coding RNAs (lncRNAs) (18,19), have emerged as central regulators of the inflammatory response (20)(21)(22)(23)(24). We recently identified lncRNAs associated with the Accepted Article © 2019 The Authors. Arthritis & Rheumatology published by Wiley Periodicals, Inc. on behalf of American College of Rheumatology inflammatory response in human OA chondrocytes that regulated the secretion of proinflammatory cytokines (22,25). Therefore, the aim of this study was to characterise the transcriptome of synovial fibroblasts isolated from obese OA and normal-weight OA patients, as well as normal-weight non-OA synovial fibroblasts by RNA sequencing, identify lncRNAs associated with the inflammatory synovial fibroblast phenotype, and to examine the expression and functional role of MALAT1, a differentially expressed lncRNA in OA synovium. Patient characteristics are shown in Table 1. Synovium was collected peri-operatively. A portion of synovium was snap-frozen in liquid nitrogen and pulverised for RNA expression analysis. The remaining synovium was used for the isolation of primary synovial fibroblasts.

RNAseq analysis
Synovial fibroblasts were cultured for 24h in 0.1% FCS culture media without antibiotics.

Principle Component Analysis and Hierarchical Clustering
The abundance of Gencode v27 defined genes in individual samples was defined as the fragments per kilobase exon per million reads mapped (FPKM) and determined using Stringtie (RNA) (see above). PCA and hierarchical clustering on Gencode v27 protein coding genes demonstrating an expression >1 FPKM was performed using Genesis (v1.7.7) (30). Data was log2 transformed following the addition of 1 FPKM. The threshold for reporting gene expression at FPKM >1 is based upon tbility to validate sequencing data using qRT-PCR (31). RNA sequencing data can be obtained at the Gene Expression Omnibus at GSEXXXXXX.

Pathway Analysis
Differentially expressed genes (>1.5 fold change, p<0.05) were analysed using DAVID and Ingenuity Pathway Analysis (IPA; www.ingenuity.com) software. In the DAVID analysis tool (https://david.ncifcrf.gov), genes were analysed using KEGG pathway option. Using IPA software, a core functional analysis was performed to identify canonical pathways and predicted upstream regulators that were significantly associated with the differentially expressed mRNAs. The significance of the association of a given canonical pathway with the differentially expressed mRNAs was measured based on the ratio of the number of mapped differentially expressed mRNAs in the data set divided by the total number of genes that map to the canonical pathway and by using the Fisher exact test to calculate a p value for the association between the mRNA and canonical pathway. For the prediction of upstream regulators, a p-value and z-score was computed based on the Accepted Article significant overlap between genes in the dataset and known targets regulated by the transcriptional regulator.

qRT-PCR
Primers for individual transcripts (Supplementary Table 1) were designed using Primer Express 3 software (Life Technologies). PCR was performed from total RNA in a onestep reaction (iTaq Universal One-Step, BioRad, UK). Relative expression was determined using the ΔΔCt method, following normalisation to 18S. RNAseq data was analysed using Cuffdiff to identify differentially expressed genes.

Synovial fibroblast proliferation
Proliferation was determined using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay kit (Promega, UK), as per the manufacturer's instructions. In brief, cells were cultured into 96-well plates and following addition of MTS reagent the absorbance at 490nm was measured using a microplate reader (SynergyHT, BioTek).

Statistical Analysis
Data were analysed using Graphpad Prism v6 by one-way ANOVA with Dunnett's Multiple Comparison Test. Data are presented as mean ± SEM, with statistical significance determined to be p<0.05.

Synovial fibroblasts isolated from obese OA patients exhibit an inflammatory phenotype
Synovial fibroblasts (n=6 patients for each group) from normal-weight non-OA, normalweight OA and obese OA were cultured for 24h and the secretion of IL-6 and CXCL8 was determined by ELISA. Compared to normal-weight non-OA, normal-weight OA fibroblasts secreted moderately (1.5-fold) more lL-6 (p<0.01) although there was no difference in the secretion of CXCL8 ( Figure 1A). However, the secretion of both IL-6 (p<0.001) and CXCL8 (p<0.05) was markedly elevated in obese OA synovial fibroblasts, compared to either normal-weight OA and non-OA fibroblasts ( Figure 1A). In addition, obese OA fibroblasts were more highly proliferative. Thus, obese OA fibroblasts exhibited a more rapid increase in cellular confluence (as determined using Incucyte) over the first 24h following passaging, compared to either normal-weight OA and non-OA fibroblasts ( Figure 1B). After 7 days culture, there were significantly greater cell numbers of obese OA fibroblasts than normal-weight OA and non-OA, as determined by MTS assay ( Figure   1C).
To further investigate the phenotype of these cells, we next isolated total RNA from the three groups of fibroblasts (n=4 patients for each group) and subjected them to 75bp,  Tables 2/3). We employed a cut-off of FPKM > 1 based upon the research by the Sequence Quality Control Consortium (31), which showed that this was the level that could be reliably confirmed by qRT-PCR. In addition, our previous studies have shown that lncRNAs and mRNAs have a mean expression level of 2.14 and 7.03, respectively (23) and that cut-offs significantly higher than > 1 FPKM would likely preclude large numbers of lncRNAs. Pathway analysis (using DAVID) of these differentially expressed mRNAs revealed that the top canonical pathways amongst downregulated genes was ECM-receptor interaction and the complement/coagulation cascade whilst for up-regulated genes this was cell-cycle, DNA replication and cytokine-cytokine interactions ( Figure 2C). This was confirmed by Ingenuity Pathway Analysis that Accepted Article identified the top pathway as cell cycle control of chromosomal replication (Supplementary Table 4) and that also identified the top upstream regulator as CDKN2, which encodes 2 proteins (p16 INK4a and p14arf) that regulate the cell cycle (Supplementary Table 5).
Comparison of normal-weight OA versus obese OA fibroblasts showed that a total of 377 mRNAs were upregulated, and 238 mRNAs were down-regulated (>2 fold change,  Table 4). We then selected key representative genes for each of the canonical pathways in Figure 2C and performed qRT-PCR to validate their differential expression between normal-weight non-OA and normal-weight OA fibroblasts (n=3 patients) or between normal-weight OA and obese OA fibroblasts using independent samples (n=4 patients). The expression of Col11A1 and CFH were confirmed to be significantly upregulated whilst the expression of CDK7, MCM6, MSH2 and CXCL6 were confirmed to be significantly downregulated in normal-weight OA fibroblasts compared to normal-weight non-OA fibroblasts. The expression of CXCL8, IL-6, CXCL5, IL-1β and CCL2 were each found to be significantly increased in obese OA fibroblasts, compared to normal-weight OA fibroblasts ( Figure   2D).

OA synovial fibroblast
We next analysed the RNAseq data to identify lncRNAs that were differentially expressed in normal-weight non-OA and obese OA fibroblasts, compared to normal-weight OA fibroblasts. Initial comparison of normal-weight OA and non-OA fibroblasts uncovered 19 lncRNAs that were differentially expressed (>2 fold change, p<0.05, change in FPKM>1) (Supplementary Table 6). This included 16 long intergenic non-coding RNA (lincRNA) and 3 antisense lncRNA, of which 10 lincRNAs were upregulated and 6 were downregulated in the normal-weight OA compared to normal-weight non-OA fibroblasts Accepted Article ( Figure 3A/B). Comparing obese OA with normal-weight OA synovial fibroblasts identified a total of 19 differentially expressed lncRNAs (Supplementary Table 6). Again, this included both antisense and lincRNAs, of which the majority were lincRNAs (15) Table 6).

Obesity-associated lincRNAs are rapidly induced in synovial fibroblasts in response to cytokine stimulation
Of the lincRNAs differentially expressed between normal-weight OA and obese OA, we

MALAT1 is differentially expressed in inflammatory OA synovial tissue
To examine the potential in vivo relevance of MALAT1, we extracted total RNA from the synovium of normal-weight non-OA (n=6), normal-weight OA (n=8) and obese OA (n=8) patients. Expression of MALAT1 was significantly upregulated in normal-weight OA synovium compared to normal-weight non-OA, and was further increased in obese OA synovium ( Figure 4A). Interestingly, down-regulation in MALAT1 expression was associated with increased expression of inflammatory genes IL-6 and CXCL8 mRNA ( Figure 4A).

MALAT1 regulates the inflammatory response and cell proliferation in obese OA
fibroblasts Subsequently, we undertook knockdown studies to ascertain whether changes in  Figure 4B) and reduced CXCL8 secretion ( Figure   4C). Fibroblasts depleted of MALAT1 also had significantly reduced cellular proliferation at 48h and 5 days, compared to fibroblasts transfected with control LNA ( Figure 4D).  Figure   5C). Further investigation using pathway analysis of the 28 differential mRNAs revealed that the most significantly affected cellular processes included "cellular growth and proliferation", whilst the most significantly affected disease functions included "inflammatory response" and "inflammatory disorders" (Figure 5D).

Discussion
Synovitis in OA has been largely understudied as a disease driver, with OA tissue often being used as a "non-inflammatory" control in comparative studies with RA. Importantly, in this paper we demonstrate for the first time that synovial fibroblasts isolated from the synovium of hip OA patients exhibit a more inflammatory and proliferative phenotype in patients who are obese, compared to normal-weight patients.
Obese OA fibroblasts secreted greater amounts of IL-6 and CXCL8 than normal-weight Adalimumab, a TNF-α neutralising antibody, was deemed ineffective at reducing disease activity in patients with erosive hand OA (32). Similarly, treatment of knee OA patients with AMG108, a neutralising IL-1 receptor antibody, failed to significantly reduce pain (33). However, these trials did not select patients based on their degree of synovial inflammation. Notably, in a clinical trial of the effectiveness of adalimumab to alleviate knee OA pain, 40% of patients had a 50% improvement in their pain score (34). Our data would suggest that the degree of synovial inflammation in the patients recruited for these studies would have been dependent on their BMI. Given our findings it would be pertinent to consider whether those patients who responded were obese and exhibited greater synovial inflammation. In addition to coding genes, our study has identified several lncRNAs that are differentially expressed in OA fibroblasts compared to non-diseased fibroblasts, as well as lncRNAs that are differentially expressed in obese OA fibroblasts compared to normalweight OA fibroblasts. Given the potential clinical implications of the inflammatory phenotype of the obese OA synovial fibroblast it was notable that the majority of the differentially expressed lncRNAs were classified as intergenic lncRNAs (lincRNAs).
LincRNAs have recently emerged as central regulators of the inflammatory response in multiple cell types. Indeed, we recently identified lincRNAs associated with the human OA chondrocyte inflammatory response, which were induced in response to an inflammatory challenge and functioned to mediate the production of IL-6 (22).
In this study we found that the expression of obesity-associated lincRNAs in synovial fibroblasts was modulated by stimulation with pro-inflammatory cytokines and adipokines.
Of the lincRNAs investigated, MALAT1 was the most responsive to pro-inflammatory challenge, being rapidly induced following stimulation of synovial fibroblasts. This to wild type mice (36). Mechanistically, MALAT1 has recently been reported to bind to (39) and modulate NFκB activity, thus regulating the LPS inflammatory response (39,40).
We also found that depletion of MALAT1 reduced the proliferation of obese OA synovial fibroblasts, suggesting that targeted inhibition of MALAT1 in the synovial joint could have It is important to note that our study has only examined the synovium from patients with hip OA. Similar to the knee, the hip is a weight-bearing joint. Therefore, the obesityassociated inflammatory synovial fibroblast reported here may be initiated and promoted by excess loading on the joint. However, obesity is associated with OA in both weightbearing and non-weight bearing joints, supporting the notion that the effect of obesity is not simply due to increased joint loading. Indeed, it is known that obesity is associated with increased circulatory levels of pro-inflammatory cytokines and adipokines (43,44).
We previously reported that the adipokine resistin is elevated systemically and more highly expressed in the synovial joint tissue in obese hip OA patients, compared to normal-weight hip OA patients (45), and it was recently found that hand OA patients exhibit increased circulatory levels of resistin (46). Thus, the obesity-associated inflammatory synovial fibroblast phenotype may be replicated in non-weight bearing OA joints.
In summary, these data demonstrate that obesity in OA patients is associated with an inflammatory synovial fibroblast phenotype, and further supports the notion that lncRNAs,