Rheumatoid Synovium Research Articles

Synovial expression of glucocorticoid receptor parallels fibroblast activation in patients with rheumatoid arthritis.

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. 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). 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. 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.

Phosphoproteomic profiling of early rheumatoid arthritis synovium reveals active signalling pathways and differentiates inflammatory pathotypes

BackgroundKinases are intracellular signalling mediators and key to sustaining the inflammatory process in rheumatoid arthritis (RA). Oral inhibitors of Janus Kinase family (JAKs) are widely used in RA, while inhibitors of other kinase families e.g. phosphoinositide 3-kinase (PI3K) are under development. Most current biomarker platforms quantify mRNA/protein levels, but give no direct information on whether proteins are active/inactive. Phosphoproteome analysis has the potential to measure specific enzyme activation status at tissue level.MethodsWe validated the feasibility of phosphoproteome and total proteome analysis on 8 pre-treatment synovial biopsies from treatment-naive RA patients using label-free mass spectrometry, to identify active cell signalling pathways in synovial tissue which might explain failure to respond to RA therapeutics.ResultsDifferential expression analysis and functional enrichment revealed clear separation of phosphoproteome and proteome profiles between lymphoid and myeloid RA pathotypes. Abundance of specific phosphosites was associated with the degree of inflammatory state. The lymphoid pathotype was enriched with lymphoproliferative signalling phosphosites, including Mammalian Target Of Rapamycin (MTOR) signalling, whereas the myeloid pathotype was associated with Mitogen-Activated Protein Kinase (MAPK) and CDK mediated signalling. This analysis also highlighted novel kinases not previously linked to RA, such as Protein Kinase, DNA-Activated, Catalytic Subunit (PRKDC) in the myeloid pathotype. Several phosphosites correlated with clinical features, such as Disease-Activity-Score (DAS)-28, suggesting that phosphosite analysis has potential for identifying novel biomarkers at tissue-level of disease severity and prognosis.ConclusionsSpecific phosphoproteome/proteome signatures delineate RA pathotypes and may have clinical utility for stratifying patients for personalised medicine in RA.

Clonal associations between lymphocyte subsets and functional states in rheumatoid arthritis synovium

Rheumatoid arthritis (RA) is an autoimmune disease involving antigen-specific T and B cells. Here, we perform single-cell RNA and repertoire sequencing on paired synovial tissue and blood samples from 12 seropositive RA patients. We identify clonally expanded CD4 + T cells, including CCL5+ cells and T peripheral helper (Tph) cells, which show a prominent transcriptomic signature of recent activation and effector function. CD8 + T cells show higher oligoclonality than CD4 + T cells, with the largest synovial clones enriched in GZMK+ cells. CD8 + T cells with possibly virus-reactive TCRs are distributed across transcriptomic clusters. In the B cell compartment, NR4A1+ activated B cells, and plasma cells are enriched in the synovium and demonstrate substantial clonal expansion. We identify synovial plasma cells that share BCRs with synovial ABC, memory, and activated B cells. Receptor-ligand analysis predicted IFNG and TNFRSF members as mediators of synovial Tph-B cell interactions. Together, these results reveal clonal relationships between functionally distinct lymphocyte populations that infiltrate the synovium of patients with RA.

Rheumatoid arthritis autologous synovial fluid affects the plasticity and function of peripheral and induced T regulatory cells in vitro

The synovial fluid (SF) microenvironment in rheumatoid arthritis (RA) may alter the stability and function of Tregs. In the present study, we assessed cytokine levels and percentage of Tregs, Tregs expressing CXCR3 (Th1-like Treg), CCR6 (Th17-like Treg) in RA peripheral blood (PB) and RA-SF using fluorescence cytometry. Effect of autologous SF on plasticity and function of RA-PB Tregs (pTregs; CD4+CD25hiCD127Lo/−) and induced vimentin-pulsed Tregs (iTregsVIM) was assessed in vitro. Cytokines and percentage of Th1-like and Th17-like Tregs were higher in RA-PB than OA-PB; higher in RA-SF than osteoarthritis (OA)-SF. Compared to OA-SF exposed OA-pTregs, RA-SF exposed RA-pTregs showed higher percentage of Th1-like (11% vs 20%) and Th17-like (16% vs 36%) Tregs; higher T-bet (p = 0.0001), RORγ (p = 0.0001) and lower FOXP3 (p = 0.0001) gene expression; and diminished percentage suppression of autologous T effector cells (36% vs 74%). RA-SF exposed iTregsVIM showed increased percentage of Th1-like and Th17-like Tregs compared to iTregsVIM exposed to AB serum (8% vs 0.1%; 21% vs 0.1%). IL-2, Tocilizumab and 5-azacytidine reduced the conversion of iTregsVIM (8% vs 2.4%; 21% vs 6.9%), when used in combination. To conclude, microenvironment in the RA synovial fluid is possibly responsible for conversion of pTregs into Th-like Tregs and their functional loss. A blockade of cytokine receptors and methyl transferases could inhibit Tregs conversion, providing clinical relevance for future Tregs targeting therapies.

Axl and MerTK regulate synovial inflammation and are modulated by IL-6 inhibition in rheumatoid arthritis

The TAM tyrosine kinases, Axl and MerTK, play an important role in rheumatoid arthritis (RA). Here, using a unique synovial tissue bioresource of patients with RA matched for disease stage and treatment exposure, we assessed how Axl and MerTK relate to synovial histopathology and disease activity, and their topographical expression and longitudinal modulation by targeted treatments. We show that in treatment-naive patients, high AXL levels are associated with pauci-immune histology and low disease activity and inversely correlate with the expression levels of pro-inflammatory genes. We define the location of Axl/MerTK in rheumatoid synovium using immunohistochemistry/fluorescence and digital spatial profiling and show that Axl is preferentially expressed in the lining layer. Moreover, its ectodomain, released in the synovial fluid, is associated with synovial histopathology. We also show that Toll-like-receptor 4-stimulated synovial fibroblasts from patients with RA modulate MerTK shedding by macrophages. Lastly, Axl/MerTK synovial expression is influenced by disease stage and therapeutic intervention, notably by IL-6 inhibition. These findings suggest that Axl/MerTK are a dynamic axis modulated by synovial cellular features, disease stage and treatment.

Computer Vision Analysis of Rheumatoid Arthritis Synovium Reveals Lymphocytic Inflammation Is Associated With Immunoglobulin Skewing in Blood.

We sought to develop computer vision methods to quantify aggregates of cells in synovial tissue and compare these with clinical and gene expression parameters. We assembled a computer vision pipeline to quantify five features encompassing synovial cell density and aggregates and compared these with pathologist scores, disease classification, autoantibody status, and RNA expression in a cohort of 156 patients with rheumatoid arthritis (RA) and 149 patients with osteoarthritis (OA). All five features were associated with pathologist scores of synovial lymphocytic inflammation (P < 0.0001). Three features that related to the cells per unit of tissue were significantly increased in patients with both seronegative and seropositive RA compared with those with OA; on the other hand, aggregate features (number and diameter) were significantly increased in seropositive, but not seronegative, RA compared with OA. Aggregate diameter was associated with the gene expression of immunoglobulin heavy-chain genes in the synovial tissue. Compared with blood, synovial immunoglobulin isotypes were skewed from IGHM and IGHD to IGHG3 and IGHG1. Further, patients with RA with high levels of lymphocytic infiltrates in the synovium demonstrated parallel skewing in their blood with a relative decrease in IGHGM (P < 0.002) and IGHD (P < 0.03) and an increase in class-switched immunoglobulin genes IGHG3 (P < 0.03) and IGHG1 (P < 0.002). High-resolution automated identification and quantification of synovial immune cell aggregates uncovered skewing in the synovium from naïve IGHD and IGHM to memory IGHG3 and IGHG1 and revealed that this process is reflected in the blood of patients with high inflammatory synovium.

Rheumatoid Synovial Fluid and Acidic Extracellular pH Modulate the Immunomodulatory Activity of Urine-Derived Stem Cells.

Urine-derived stem cells (UdSCs) possess a remarkable anti-inflammatory and immune-modulating activity. However, the clinical significance of UdSCs in autoimmune inflammatory diseases such as rheumatoid arthritis (RA) is yet to be explored. Hence, we tested the UdSCs response to an articular RA microenvironment. To simulate the inflamed RA joint more authentically in vitro, we treated cells with rheumatoid synovial fluids (RASFs) collected from RA patients, serum deprivation, acidosis (pH 7.0 and 6.5), and their combinations. Firstly, the RASFs pro-inflammatory status was assessed by cytokine quantification. Then, UdSCs were exposed to the RA environmental factors for 48 h and cell proliferation, gene expression and secretion of immunomodulatory factors were evaluated. The immunosuppressive potential of pre-conditioned UdSCs was also assessed via co-cultivation with activated peripheral blood mononuclear cells (PBMCs). In all experimental conditions, UdSCs' proliferation was not affected. Conversely, extracellular acidosis considerably impaired the viability/proliferation of adipose tissue-derived stem cells (ATSCs). In the majority of cases, exposure to RA components led to the upregulated expression of IL-6, TSG6, ICAM-1, VCAM-1, and PD-L1, all involved in immunomodulation. Upon RASFs and acidic stimulation, UdSCs secreted higher levels of immunomodulatory cytokines: IL-6, IL-8, MCP-1, RANTES, GM-CSF, and IL-4. Furthermore, RASFs and combined pretreatment with RASFs and acidosis promoted the UdSCs-mediated immunosuppression and the proliferation of activated PBMCs was significantly inhibited. Altogether, our data indicate that the RA microenvironment certainly has the capacity to enhance UdSCs' immunomodulatory function. For potential preclinical/clinical applications, the intra-articular injection might be a reasonable approach to maximize UdSCs' therapeutic efficiency in the RA treatment.

Rheumatoid arthritis synovial fluid shows enrichment of T-cells producing GMCSF which are polyfunctional for TNFα and IFNγ.

GMCSF+T-cells may be involved in pathogenesis of rheumatoid arthritis (RA), and polyfunctionality may be a marker of pathogenicity. Although, higher frequencies of CD4+GMCSF+ T-cells have been reported, there are no data on CD8+GMCSF+ T-cells or polyfunctionality.Our objective was to enumerate frequencies of CD8+GMCSF+ T cells in RA blood and synovial fluid (SF), and assess their polyfunctionality, memory phenotype and cytotoxic ability. This study included RA patients (blood samples,in some with paired synovial fluid (SF)), healthy controls (HC) (blood) and SpA patients (SF). In some RA patients' blood was sampled twice, before and 16-24 weeks after methotrexate (MTX) treatment. After mononuclear cell isolation from blood and SF, ex-vivo stimulation using PMA/Ionomycin was done, and cells were stained (surface and intracellular after permeabilisation/fixation). Subsequently, frequencies of GMCSF+CD8+ and CD4+ T-cells, polyfunctionality (TNFα, IFNγ, IL-17), phenotype (memory) and perforin/granzyme expression were assessed by flowcytometry. There was no significant difference in frequencies of GMCSF+CD8+ (3.7, 4.1%, p=0.540) or GMCSF+CD4+ T-cells (4.5, 5.2%, p=0.450) inblood of RA and HC. However, there was significant enrichment of both CD8+GMCSF+ (5.8, 3.9%, p=0.0045) and CD4+GMCSF+ (8.5, 4.5%, p=0.0008) T-cells inSF compared to blood in RA patients. Polyfunctional triple cytokine positive TNFα+IFNγ+GMCSF+CD8+T-cells (81, 36%, p=0.049) and CD4+T-cells (48, 32%, p=0.010) was also higher in SF compared to blood in RA. CD8+ T cells showed higher frequency of effector-memory phenotype and granzyme-B expression in RA-SF. On longitudinal follow-up, blood CD4+GMCSF+ T-cells significantly declined (4.6, 2.9%, p=0.0014) post-MTX. We report a novel finding of enrichment of CD8+GMCSF+ in addition to CD4+GMCSF+ T-cells in RA-SF. These cells showed higher polyfunctionality for TNFα and IFNγ, and effector memory phenotype suggesting their involvement in RA pathogenesis.

Inhibition of Cdc37 Ameliorates Arthritis in Collagen-Induced Arthritis Rats by Inhibiting Synoviocyte Proliferation and Migration Through the ERK Pathway.

Rheumatoid arthritis (RA) is a chronic autoimmune disease that can lead to synovial inflammation, pannus formation, cartilage damage, bone destruction, and ultimate disability. Fibroblast-like synoviocytes (FLS) are involved in the pathogenetic mechanism of RA. Cdc37 (cell division cycle protein 37) is regarded as a molecular chaperone involved in various physiological processes such as cell cycle progression, cell proliferation, cell signal transduction, tumorigenesis, and progression. However, the precise role of Cdc37 in the pathogenesis of rheumatoid arthritis (RA) remains uncertain. In our study, we found that Cdc37 expression was upregulated in human rheumatoid synovia in contrast with the normal group. Interestingly, Cdc37 activated the ERK pathway to promote RA-FLS proliferation and migration in vitro. Ultimately, in vivo experiments revealed that silencing of Cdc37 alleviated ankle swelling and cartilage destruction and validated the ERK signaling pathways in vitro findings. Collectively, we demonstrate that Cdc37 promotes the proliferation and migration of RA-FLS by activation of ERK signaling pathways and finally aggravates the progression of RA. These data indicated that Cdc37 may be a novel target for the treatment of RA.

S100A8 alarmin supports IL-6 and metalloproteinase-9 production by fibroblasts in the synovial microenvironment of peripheral spondyloarthritis.

Spondyloarthritis (SpA) is a common autoinflammatory disease. S100A8/ S100A9 alarmin is strongly expressed in the synovial sublining layers of psoriatic arthritis. S100A8/ S100A9 is the most abundant protein in rheumatoid arthritis synovial fluid (SF) and has a key role in promoting IL-6 expression in fibroblast-like synoviocytes (FLS). The molecular mechanisms and the role of S100-alarmins in the synovial microenvironment of SpA have never been demonstrated. Here, we confirm the effect of the synovial microenvironment of peripheral SpA on interleukin-6 (IL-6) and metalloproteinase (MMP)-9 production by FLS. MMP-9 expression and activity were detected, which were reduced in the presence of anti-IL-6R. Analyzing cell signaling mechanisms, we found that stimulation with IL-6 co-triggered MMP-9 and IL-10 secretion. MMP-9 secretion depended on JNK and p38 MAPKs, whereas IL-10 secretion was dependent on the JAK pathway as a potential feedback mechanism controlling IL-6-induced MMP-9 expression. Using a proteomic approach, we identified S100A8 in the peripheral SpA SF. This presence was confirmed by immunoblotting. S100A8 increased the IL-6 secretion via ERK and p38 MAPK pathways. Furthermore, anti-S100A8/A9 reduced both IL-6 and MMP-9 production induced by SpA SF in FLS. Our data reveal a marked relationship between S100A8 alarmin with IL-6 and MMP-9 secretion by FLS in the real synovial microenvironment of peripheral SpA. These results identify a mechanism linking S100A8 to the pathogenesis of peripheral SpA.

Reciprocal Alterations in Osteoprogenitor and Immune Cell Populations in Rheumatoid Synovia.

Rheumatoid arthritis (RA) is chronic, autoimmune joint inflammation characterized by irreversible joint destruction. Besides increased resorption, destruction is a result of decreased bone formation, due to suppressed differentiation and function of the mesenchymal lineage-derived osteoblasts in inflammatory milieu. In this study, we analyzed the cellular composition of synovial tissue from 11 RA and 10 control patients harvested during planned surgeries in order to characterize resident synovial progenitor populations. Synovial cells were released by collagenase, and labeled for flow cytometry by two antibody panels: 1. CD3-FITC, CD14-PE, 7-AAD, CD11b-PECy7, CD235a-APC, CD19-APCeF780; and 2. 7-AAD, CD105-PECy7, CD45/CD31/CD235a-APC, and CD200-APCeF780. The proportions of lymphocytes (CD3+, CD19+) and myeloid (CD11b+, CD14+) cells were higher in synovial tissue from the patients with RA than in the controls. Among non-hematopoietic (CD45−CD31−CD235a−) cells, there was a decrease in the proportion of CD200+CD105− and increase in the proportion of CD200−CD105+ cells in synovial tissue from the patients with RA in comparison to the control patients. The proportions of both populations were associated with inflammatory activity and could discriminate between the RA and the controls.

Histopathology of Psoriatic Arthritis Synovium-A Narrative Review.

Psoriatic arthritis (PsA) is a phenotypically heterogeneous chronic inflammatory disease associated to type I major histocompatibility complex alleles whose complex pathogenesis is still not completely understood. The psoriatic synovium shares general features of chronic inflammation with rheumatoid arthritis (RA) and other arthritis, such as hyperplasia of the intimal lining layer, sublining influx of inflammatory cells and neoangiogenesis, but recognizing disease-specific histopathologic findings may help in diagnosis and definition of therapeutic targets. Available literature reports conflicting data regarding the extension of lining hyperplasia, that does not allow depiction from RA. Sublining inflammatory cells consist of T and B cells and macrophages, plasma cells, mast cells and follicular dendritic cells, with a higher amount of overall T, mast cell and IL-17 producing CD8+ T lymphocytes and lower proportion of plasma cells when compared to the rheumatoid synovium. The amount of synovium IL17+ CD8+ T cells correlates positively to measures of disease activity. Lymphoid follicles with characteristics of germinal centers have been identified, similar to the ones described in RA. Neoangiogenesis is more prominent in PsA but can also be an outstanding feature in some RA samples, and different molecules involved in the process appear to have different influence in each disease. IL-17 and IL-22 expression in the synovium does not allow depiction between diseases. Among other cytokines and molecules likely implicated in disease physiopathology, only IL-35 is demonstrated to be reduced in PsA when compared to RA.

Preliminary Report: Osteoarthritis and Rheumatoid Arthritis Synovial Fluid Increased Osteoclastogenesis In Vitro by Monocyte Differentiation Pathway Regulating Cytokines.

Background Rheumatoid arthritis (RA) and osteoarthritis (OA) are common joint diseases associated with changes in local, as well as systemic bone structure and osteoclast function. We investigated how the different soluble inflammatory stimuli in these diseases can affect osteoclastogenesis and bone resorption in vitro. Methods. Human peripheral blood mononuclear cell-derived osteoclasts were cultured on bone slices with serum from treatment-naïve RA patients and healthy controls and with synovial fluid samples acquired from RA and OA patients. The concentrations of 29 different cytokines and related proteins, including RANKL and OPG, were analyzed in the fluids tested. Results RA serum and synovial fluid increased both osteoclastogenesis and bone resorption. Osteoclastogenesis and activity increased more in the cultures containing OA than RA synovial fluid. The osteoclasts cultured in different culture media exhibited different phenotypes, especially the cells cultured with OA synovial fluid were generally larger and had more nuclei. A general increase in proinflammatory cytokines in RA synovial fluid and serum was found. Surprisingly, OA synovial fluid showed lower levels of osteoclastogenesis inhibiting cytokines, such as IL-4 and IL-10, than RA synovial fluid, which at least partly explains more pronounced osteoclastogenesis. No significant difference was found in RANKL or OPG levels. Conclusion The proinflammatory stimulus in OA and RA drives the monocyte differentiation towards inflammatory osteoclastogenesis and altered osteoclast phenotype.

Utility of Baseline Transcriptomic Analysis of Rheumatoid Arthritis Synovium as an Indicator for Long-Term Clinical Outcomes.

Objective:Rheumatoid arthritis is a chronic inflammatory autoimmune disease that can lead to synovial damage, persistent joint pain, and functional disability. Our objective was to evaluate baseline synovial transcriptome from early inflammatory arthritis patients (EIA) and identify pretreatment biomarkers that could potentially provide insights into long-term functional outcomes of rheumatoid arthritis (RA).MethodsSynovial biopsies from clinically inflamed knee joints were procured from either 17 EIA patients before initiation of disease modifying anti-rheumatic drug (DMARD) therapy (DMARD-naïve EIA) using the minimally invasive closed needle biopsy technique or advanced RA patients undergoing arthroplasty. Affymetrix Human Genome U133 Plus 2 microarray platform was used to profile the synovial transcriptome. The cohort was followed clinically for a median of 12.3 years, and patient data was collected at each visit. Short-term and long-term clinical outcomes were determined by assessing RA-associated clinical parameters Statistical adjustments were made to account for asynchronous clinical visits and duration of follow up.ResultsBased on the transcriptomic analysis, we identified 5 differentially expressed genes (DEGs), including matrix metalloproteinase (MMP)-1 (fibroblast collagenase) and MMP-3 (stromelysin-1) in DMARD-naïve EIA patients, relative to advanced RA patients (q < 0.05). Dichotomous expression of MMP-1 and MMP-3 mRNA and protein was confirmed by qPCR and immunohistochemistry respectively, based on which DMARD-naïve EIA subjects were classified as MMP-high or MMP-low. Hierarchical clustering of transcriptomic data identified 947 DEGs between MMP-high and MMP-low cohorts. Co-expression and IPA analysis of DEGs in the MMP-high cohort showed an enrichment of genes that participated in metabolic or biochemical functions and intracellular immune signaling were regulated through NF-κB and β-catenin complexes and correlated with markers of systemic inflammation. Analysis of short-term clinical outcomes in MMP-high cohort showed a significant reduction in the DAS-CRP scores relative to baseline (P <0.001), whereas area under the curve analyses of modified HAQ (mHAQ) scores correlated negatively with baseline MMP-1 (R = −0.59, P = 0.03). Further, longitudinal mHAQ scores, number of swollen joints, number of DMARDs and median follow-up duration appeared to be higher in MMP-low cohort.ConclusionOverall, our results indicate that the gene expression profiling of synovial biopsies obtained at the DMARD-naive stage in patients with EIA categorizes them into subsets with varying degrees of inflammation and can predict the future of long-term clinical outcome.

Three-dimensional spatial transcriptomics uncovers cell type localizations in the human rheumatoid arthritis synovium

The inflamed rheumatic joint is a highly heterogeneous and complex tissue with dynamic recruitment and expansion of multiple cell types that interact in multifaceted ways within a localized area. Rheumatoid arthritis synovium has primarily been studied either by immunostaining or by molecular profiling after tissue homogenization. Here, we use Spatial Transcriptomics, where tissue-resident RNA is spatially labeled in situ with barcodes in a transcriptome-wide fashion, to study local tissue interactions at the site of chronic synovial inflammation. We report comprehensive spatial RNA-Seq data coupled to cell type-specific localization patterns at and around organized structures of infiltrating leukocyte cells in the synovium. Combining morphological features and high-throughput spatially resolved transcriptomics may be able to provide higher statistical power and more insights into monitoring disease severity and treatment-specific responses in seropositive and seronegative rheumatoid arthritis.

Identification of T Peripheral Helper (Tph) Cells.

T peripheral helper (Tph) cells represent a T cell population that stimulates B cell responses within peripheral tissues. Like T follicular helper (Tfh) cells, PD-1hi CXCR5- CD4+ Tph cells produce IL-21 and CXCL13, yet these cells differ from Tfh cells in expression of both transcription factors and chemokine receptors. Originally identified in rheumatoid arthritis synovium, Tph cells are expanded in multiple autoimmune diseases. Tph cells can be identified and distinguished from Tfh cells by both flow cytometry and mass cytometry. Here, we describe detailed methods to identify Tph cells and characterize associated phenotypic features and functions. These protocols and additional notes provide a guide to identify and explore the roles of Tph cells in various human diseases.

CYT387 Inhibits the Hyperproliferative Potential of Fibroblast-like Synoviocytes via Modulation of IL-6/JAK1/STAT3 Signaling in Rheumatoid Arthritis

ABSTRACT Fibroblast-like synoviocytes (FLS) are the critical effector cells primarily involved in rheumatoid arthritis (RA) disease pathogenesis. Interleukin (IL)-6, a proinflammatory cytokine most abundantly expressed in the rheumatoid synovium, promotes Janus kinase (JAK)/signal transducer and transcriptional activator (STAT) signaling cascade activation in RA-FLS, thus leading to its aggressive phenotype, invasiveness, and joint destruction. Momelotinib (CYT387) is a selective small-molecule inhibitor of JAK1/2 and is clinically approved to treat myelofibrosis. However, the therapeutic efficacy of CYT387 in FLS mediated RA pathogenesis is less known. In the present study, we investigated the modulatory effect of CYT387 on IL6/JAK/STAT signaling cascade in FLS induced RA pathogenesis. CYT387 treatment inhibited IL-6 induced high proliferative and migratory potential of FLS cells isolated from adjuvant-induced arthritic (AA) rats. CYT387 reduced the expression of PRMT5, survivin, and HIF-1α mediated by IL-6/sIL-6R in AA-FLS in a dose-dependent manner. The IL-6/sIL-6R induced expression of angiogenic factors such as VEGF and PIGF in AA-FLS cells was found downregulated by CYT387 treatment. Importantly, CYT387 significantly reduced IL-6/sIL-6R dependent activation of JAK1 and STAT3 and increased SOCS3 expression in AA-FLS cells. Next, the S3I-201 mediated blockade of STAT3 activation supported the inhibitory effect of CYT387 on IL-6/JAK1/STAT3 signaling cascade in AA-FLS. Overall, this study proves that CYT387 inhibits proliferation, migration, and pathogenic disease potential of FLS isolated from adjuvant-induced arthritic (AA) rats via targeting IL-6/JAK1/STAT3 signaling cascade.

Analysis of DIP2C as a novel regulator for epithelial-mesenchymal transition of rheumatoid arthritis synovium and a potential therapeutic target

Rheumatoid arthritis (RA) is an autoimmune disease that can cause damage to the joints, cartilage and bone. There is no cure but early diagnosis can help mitigate damage. Sometimes RA is particularly difficult to treat, for example when the disease took a long time to be diagnosed. Associate Professor Masao Tanaka, Graduate School of Medicine, Kyoto University, Japan, leads a team of researchers working to improve understanding of the causes of poor response to treatment in RA with a long morbidity. The goal is to restore patients' therapeutic responsiveness, thereby improving outcomes. A previous focus for Tanaka was on a protein called FSTL1. He is now exploring DIP2 as a binding molecule for FSTL1. Other important mechanisms Tanaka is exploring are DNA methylation and the mechanisms of carnitine, which has been found to decrease a variety of activation signalling by inhibiting ceramide production in T cells. He and the team are exploring the involvement of these mechanisms in DIP2. In his most recent investigations, Tanaka is exploring DIP2C as a novel regulator for epithelial-mesenchymal transition of RA synovium and a potential therapeutic target. He is focusing on molecules that are expressed in the cells in joints, making the work directly applicable to RA. The team is carrying out a cohort study called KURAMA (Kyoto University Rheumatoid Arthritis Management Alliance) that involves around 2,000 outpatients with RA. Ultimately, Tanaka hopes to identify a reproducible combination of patient conditions and therapeutic interventions that achieve better treatment results for RA patients.

Interleukin-7: a potential factor supporting B-cell maturation in the rheumatoid arthritis synovium

The exact function of interleukin-7 (IL-7) in autoimmune diseases remains unclear although it is a recognised therapeutic target for cytokine blockade. Our objective was to investigate the regulation and downstream effect of IL-7 in diseased tissue from rheumatoid arthritis (RA) patients notably with respect to its function as bone turnover regulator and tissue architecture (TA) organiser. Synovial tissues (fresh, frozen or xed) were obtained from our tissue bank and distributed between experiments for live cell cultures, histology, immunohistochemistry or gene expression array by qPCR. IL-7 expression in synoviocyte cultures was up-regulated by pro-in ammatory cytokines, notably IL-6. Gene expression pro ling segregated synovial biopsies based on the presence of B/plasma cells and ectopic TA. IL-7 gene expression was associated with that of several genes whose function was to support B-cell maturation in tissue with distinct B-cell aggregates (despite the lack of IL-7-Receptor expression on B-cells) as well as with ectopic germinal-like centres. IL-7 was associated with bone turnover regulation in biopsies with diffuse in ltration. A novel relationship between the IL-7 and IL-6 axis was also highlighted in human tissue. Overall, IL-7 may contribute to the maintenance of the pro-in ammatory cycle perpetuating in ammation in RA synovium. We therefore propose a novel role for IL-7 as an orchestrator of TA with an impact on B-cell maturation in relation with IL-6.

Novel insights into macrophage diversity in rheumatoid arthritis synovium.

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease affecting joints and causing progressive damage and disability. Macrophages are of critical importance in the initiation and perpetuation of synovitis in RA, they can function as antigen presenting cells leading to T-cell dependent B-cell activation, assume a variety of inflammatory cell states with the production of destructive cytokines, but also contribute to tissue homeostasis/repair. The recent development of high-throughput technologies, including bulk and single cells RNA-sequencing, has broadened our understanding of synovial cell diversity, and opened novel perspectives to the discovery of new potential therapeutic targets in RA. In this review, we will focus on the relationship between the synovial macrophage infiltration and clinical disease severity and response to treatment. We will then provide a state-of-the-art picture of the biological roles of synovial macrophages and distinct macrophage subsets described in RA. Finally, we will review the effects of approved conventional and biologic drugs on the synovial macrophage component and highlight the therapeutic potential of future strategies to re-program macrophage phenotypes in RA.