Jason D. Turner

Nonclassical Ly6C(-) Monocytes Drive the Development of Inflammatory Arthritis in Mice

Different subsets and/or polarized phenotypes of monocytes and macrophages may play distinct roles during the development and resolution of inflammation. Here, we demonstrate in a murine model of rheumatoid arthritis that nonclassical Ly6C(-) monocytes are required for the initiation and progression of sterile joint inflammation. Moreover, nonclassical Ly6C(-) monocytes differentiate into inflammatory macrophages (M1), which drive disease pathogenesis and display plasticity during the resolution phase. During the development of arthritis, these cells polarize toward an alternatively activated phenotype (M2), promoting the resolution of joint inflammation. The influx of Ly6C(-) monocytes and their subsequent classical and then alternative activation occurs without changes in synovial tissue-resident macrophages, which express markers of M2 polarization throughout the course of the arthritis and attenuate joint inflammation during the initiation phase. These data suggest that circulating Ly6C(-) monocytes recruited to the joint upon injury orchestrate the development and resolution of autoimmune joint inflammation.

The role of the synovial fibroblast in rheumatoid arthritis pathogenesis.

Purpose of reviewSynovial fibroblasts continue to grow in prominence both as the subjects of research into the pathogenesis of rheumatoid arthritis and as novel therapeutic targets. This timely review aims to integrate the most recent findings with existing paradigms of fibroblast-related mechanisms of disease. Recent findingsLinking the role of synovial fibroblasts as innate sentinels expressing pattern recognition receptors such as toll-like receptors to their effector roles in joint damage and interactions with leukocyte subpopulations has continued to advance. Understanding of the mechanisms underlying increased fibroblast survival in the inflamed synovium has led to therapeutic strategies such as cyclin-dependent kinase inhibition. Major advances have taken place in understanding of the interactions between epigenetic and micro-RNA regulation of transcription in synovial fibroblasts, improving our understanding of the unique pathological phenotype of these cells. Finally, the impact of new markers for fibroblast subpopulations is beginning to become apparent, offering the potential for targeting of pathological cells as the roles of different populations become clearer. SummaryOver the past 2 years, major advances have continued to emerge in understanding of the relationship between synovial fibroblasts and the regulation of inflammatory pathways in the rheumatoid arthritis synovium.

Rheumatoid synovial fibroblasts differentiate into distinct subsets in the presence of cytokines and cartilage

BackgroundWe investigated two distinct synovial fibroblast populations that were located preferentially in the lining or sub-lining layers and defined by their expression of either podoplanin (PDPN) or CD248, and explored their ability to undergo self-assembly and transmigration in vivo.MethodsSynovial fibroblasts (SF) were cultured in vitro and phenotypic changes following stimulation with interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and transforming growth factor (TGF)-β1 were examined. To examine the phenotype of SF in vivo, a severe combined immunodeficiency (SCID) human-mouse model of cartilage destruction was utilised.ResultsSF in the lining layer in rheumatoid arthritis (RA) expressed high levels of PDPN compared to the normal synovium, whereas CD248 expression was restricted to sub-lining layer cells. TNF-α or IL1 stimulation in vitro resulted in an increased expression of PDPN. In contrast, stimulation with TGF-β1 induced CD248 expression. In the SCID human-mouse model, rheumatoid SF recapitulated the expression of PDPN and CD248. Fibroblasts adjacent to cartilage expressed PDPN, and attached to, invaded, and degraded cartilage. PDPN+ CD248– SF preceded the appearance of PDPN– CD248+ cells in contralateral implants.ConclusionsWe have identified two distinct SF populations identified by expression of either PDPN or CD248 which are located within different anatomical compartments of the inflamed synovial membrane. These markers discriminate between SF subsets with distinct biological properties. As PDPN-expressing cells are associated with early fibroblast migration and cartilage erosion in vivo, we propose that PDPN-expressing cells may be an attractive therapeutic target in RA.

Treatment of inflammatory arthritis via targeting of tristetraprolin, a master regulator of pro-inflammatory gene expression.

Objectives Tristetraprolin (TTP), a negative regulator of many pro-inflammatory genes, is strongly expressed in rheumatoid synovial cells. The mitogen-activated protein kinase (MAPK) p38 pathway mediates the inactivation of TTP via phosphorylation of two serine residues. We wished to test the hypothesis that these phosphorylations contribute to the development of inflammatory arthritis, and that, conversely, joint inflammation may be inhibited by promoting the dephosphorylation and activation of TTP. Methods The expression of TTP and its relationship with MAPK p38 activity were examined in non-inflamed and rheumatoid arthritis (RA) synovial tissue. Experimental arthritis was induced in a genetically modified mouse strain, in which endogenous TTP cannot be phosphorylated and inactivated. In vitro and in vivo experiments were performed to test anti-inflammatory effects of compounds that activate the protein phosphatase 2A (PP2A) and promote dephosphorylation of TTP. Results TTP expression was significantly higher in RA than non-inflamed synovium, detected in macrophages, vascular endothelial cells and some fibroblasts and co-localised with MAPK p38 activation. Substitution of TTP phosphorylation sites conferred dramatic protection against inflammatory arthritis in mice. Two distinct PP2A agonists also reduced inflammation and prevented bone erosion. In vitro anti-inflammatory effects of PP2A agonism were mediated by TTP activation. Conclusions The phosphorylation state of TTP is a critical determinant of inflammatory responses, and a tractable target for novel anti-inflammatory treatments.

Functionally distinct disease-associated fibroblast subsets in rheumatoid arthritis

Fibroblasts regulate tissue homeostasis, coordinate inflammatory responses, and mediate tissue damage. In rheumatoid arthritis (RA), synovial fibroblasts maintain chronic inflammation which leads to joint destruction. Little is known about fibroblast heterogeneity or if aberrations in fibroblast subsets relate to pathology. Here, we show functional and transcriptional differences between fibroblast subsets from human synovial tissues using bulk transcriptomics of targeted subpopulations and single-cell transcriptomics. We identify seven fibroblast subsets with distinct surface protein phenotypes, and collapse them into three subsets by integrating transcriptomic data. One fibroblast subset, characterized by the expression of proteins podoplanin, THY1 membrane glycoprotein and cadherin-11, but lacking CD34, is threefold expanded in patients with RA relative to patients with osteoarthritis. These fibroblasts localize to the perivascular zone in inflamed synovium, secrete proinflammatory cytokines, are proliferative, and have an in vitro phenotype characteristic of invasive cells. Our strategy may be used as a template to identify pathogenic stromal cellular subsets in other complex diseases.Synovial fibroblasts are thought to be central mediators of joint destruction in rheumatoid arthritis (RA). Here the authors use single-cell transcriptomics and flow cytometry to identify synovial fibroblast subsets that are expanded and display distinct tissue distribution and function in patients with RA.

Stromal cell markers are differentially expressed in the synovial tissue of patients with early arthritis

Introduction Previous studies have shown increased expression of stromal markers in synovial tissue (ST) of patients with established rheumatoid arthritis (RA). Here, ST expression of stromal markers in early arthritis in relationship to diagnosis and prognostic outcome was studied. Methods ST from 56 patients included in two different early arthritis cohorts and 7 non-inflammatory controls was analysed using immunofluorescence to detect stromal markers CD55, CD248, fibroblast activation protein (FAP) and podoplanin. Diagnostic classification (gout, psoriatic arthritis, unclassified arthritis (UA), parvovirus associated arthritis, reactive arthritis and RA), disease outcome (resolving vs persistent) and clinical variables were determined at baseline and after follow-up, and related to the expression of stromal markers. Results We observed expression of all stromal markers in ST of early arthritis patients, independent of diagnosis or prognostic outcome. Synovial expression of FAP was significantly higher in patients developing early RA compared to other diagnostic groups and non-inflammatory controls. In RA FAP protein was expressed in both lining and sublining layers. Podoplanin expression was higher in all early inflammatory arthritis patients than controls, but did not differentiate diagnostic outcomes. Stromal marker expression was not associated with prognostic outcomes of disease persistence or resolution. There was no association with clinical or sonographic variables. Conclusions Stromal cell markers CD55, CD248, FAP and podoplanin are expressed in ST in the earliest stage of arthritis. Baseline expression of FAP is higher in early synovitis patients who fulfil classification criteria for RA over time. These results suggest that significant fibroblast activation occurs in RA in the early window of disease.

Pathologically distinct fibroblast subsets drive inflammation and tissue damage in arthritis

The identification of lymphocyte subsets with non-overlapping effector functions has been pivotal to the development of targeted therapies in immune mediated inflammatory diseases (IMIDs). However it remains unclear whether fibroblast subclasses with non-overlapping functions also exist and are responsible for the wide variety of tissue driven processes observed in IMIDs such as inflammation and damage. Here we identify and describe the biology of distinct subsets of fibroblasts responsible for mediating either inflammation or tissue damage in arthritis. We show that deletion of FAPα+ synovial cells suppressed both inflammation and bone erosions in murine models of resolving and persistent arthritis. Single cell transcriptional analysis identified two distinct fibroblast subsets: FAPα+ THY1+ immune effector fibroblasts located in the synovial sub-lining, and FAPα+ THY1- destructive fibroblasts restricted to the synovial lining. When adoptively transferred into the joint, FAP α+ THY1- fibroblasts selectively mediate bone and cartilage damage with little effect on inflammation whereas transfer of FAP α+ THY1+ fibroblasts resulted in a more severe and persistent inflammatory arthritis, with minimal effect on bone and cartilage. Our findings describing anatomically discrete, functionally distinct fibroblast subsets with non-overlapping functions have important implications for cell based therapies aimed at modulating inflammation and tissue damage.

High dimensional analyses of cells dissociated from cryopreserved synovial tissue

Background Detailed molecular analyses of cells from rheumatoid arthritis (RA) synovium hold promise in identifying cellular phenotypes that drive tissue pathology and joint damage. The Accelerating Medicines Partnership (AMP) RA/SLE network aims to deconstruct autoimmune pathology by examining cells within target tissues through multiple high-dimensional assays. Robust standardized protocols need to be developed before cellular phenotypes at a single cell level can be effectively compared across patient samples. Methods Multiple clinical sites collected cryopreserved synovial tissue fragments from arthroplasty and synovial biopsy in a 10%-DMSO solution. Mechanical and enzymatic dissociation parameters were optimized for viable cell extraction and surface protein preservation for cell sorting and mass cytometry, as well as for reproducibility in RNA sequencing (RNA-seq). Cryopreserved synovial samples were collectively analyzed at a central processing site by a custom-designed and validated 35-marker mass cytometry panel. In parallel, each sample was flow sorted into fibroblast, T cell, B cell, and macrophage suspensions for bulk population RNA-seq and plate-based single cell CEL-Seq2 RNA-seq. Results Upon dissociation, cryopreserved synovial tissue fragments yielded a high frequency of viable cells, comparable to samples undergoing immediate processing. Optimization of synovial tissue dissociation across six clinical collection sites with ∼30 arthroplasty and ∼20 biopsy samples yielded a consensus digestion protocol using 100µg/mL of Liberase TL™ enzyme. This protocol yielded immune and stromal cell lineages with preserved surface markers and minimized variability across replicate RNA-seq transcriptomes. Mass cytometry analysis of cells from cryopreserved synovium distinguished: 1) diverse fibroblast phenotypes, 2) distinct populations of memory B cells and antibody-secreting cells, and 3) multiple CD4+ and CD8+ T cell activation states. Bulk RNA sequencing of sorted cell populations demonstrated robust separation of synovial lymphocytes, fibroblasts, and macrophages. Single cell RNA-seq produced transcriptomes of over 1000 genes/cell, including transcripts encoding characteristic lineage markers identified. Conclusion We have established a robust protocol to acquire viable cells from cryopreserved synovial tissue with intact transcriptomes and cell surface phenotypes. A centralized pipeline to generate multiple high-dimensional analyses of synovial tissue samples collected across a collaborative network was developed. Integrated analysis of such datasets from large patient cohorts may help define molecular heterogeneity within RA pathology and identify new therapeutic targets and biomarkers.

THU0055 An anatomically distinct pathogenic fibroblast subset drives inflammation in arthritis

Background Fibroblasts are key effector cells in the persistence of synovial inflammation and joint damage. It is not yet known whether specific subsets of synovial fibroblasts exist, and if so, if they are responsible for the distinct fibroblast mediated features observed in inflammatory arthritis, such as invasion of cartilage, bone damage, and persistence of inflammation. Objectives Here we identify and describe the biology of a functionally distinct pathogenic fibroblast cell type marked by the co-expression of Thy1.2 and Podoplanin (Pdpn), that is responsible for persistence of synovial inflammation. Methods We used the serum transfer arthritis (STA) model to induce joint inflammation. To identify putative subsets of fibroblasts we used flow cytometry using established markers of stromal cells, on enzymatically digested synovial tissue. These cells were localised in tissue sections using immunohistochemistry or immunofluorescence staining. Proliferation of subsets in vivo during the time course of STA was determined by BrdU incorporation studies. Transcriptomic analysis was performed using ultra-low input RNA sequencing on flow sorted populations of cells and single cell analysis performed on CD45 negative gated cells. Results Using FAP as a biomarker of activated synovial fibroblasts we have been able to identify and define distinct subsets of synovial fibroblasts based on their co-expression of Thy1.2 and Pdpn. These subsets reside in distinct compartments of the synovial microanatomy including the lining layer (LL), sub-lining layer (SL) and a subset of pericytes. We found that FAP +cells within the SL are highly proliferative and their expansion in cell number positively correlates with inflammation. Global deletion of FAP +mesenchymal cells in the synovium using a FAP-DTR mouse attenuated synovial inflammation; protected against erosive bone damage and lead to reduced leucocyte accumulation as a result of reduced chemokine and cytokine production by synovial fibroblasts within the membrane. Collectively these data suggest a pathogenic pro-inflammatory role for these cells. Transcriptomic analysis by both ultra-low input bulk RNA sequencing and single cell analysis revealed these subsets to be transcriptionally distinct with the greatest transcriptional differences observed between LL and SL cells, demonstrating a site specific transcriptional program for cells within these compartments. Gene signature analysis of SL FAP +cells was consistent with an immune effector cell phenotype, in contrast to lining layer cells that express genes associated with matrix remodelling. Finally, to test this hypothesis we injected FAP +LL cells and FAP +SL cells into inflamed ankle joints of mice. The injection of FAP +SL cells lead to more severe and prolonged joint inflammation, whereas injection of LL FAP +cells had no effect. Finally, we identified these cell subsets within human synovial tissue and have demonstrated the expansion of SL cells with inflammation. Conclusions Synovial inflammation is associated with the expansion, activation and differentiation of fibroblasts into distinct functional subsets of cells that regulate those specific aspects of inflammatory joint pathology. Direct targeting of specific pathogenic subsets of synovial fibroblasts may provide a novel, non-immunosuppressive approach to the treatment of inflammatory arthritis. Disclosure of Interest None declared

Single Cell Transcriptomics And Flow Cytometry Reveal Disease-Associated Fibroblast Subsets In Rheumatoid Arthritis

Fibroblasts mediate normal tissue matrix remodeling, but they can cause fibrosis or tissue destruction following chronic inflammation. In rheumatoid arthritis (RA), synovial fibroblasts expand, degrade cartilage, and drive joint inflammation. Little is known about fibroblast heterogeneity or if aberrations in fibroblast subsets relate to disease pathology. Here, we used an integrative strategy, including bulk transcriptomics on targeted subpopulations and unbiased single-cell transcriptomics, to analyze fibroblasts from synovial tissues. We identify 7 phenotypic fibroblast subsets with distinct surface protein phenotypes, and these collapsed into 3 subsets based on transcriptomics data. One subset expressing PDPN, THY1, but lacking CD34 was 3-fold expanded in RA relative to osteoarthritis (P=0.007); most of these cells expressed CDH11. The subsets were found to differ in expression of cytokines and matrix metalloproteinases, localization in synovial microanatomy, and in response to TNF. Our approach provides a template to identify pathogenic stromal cellular subsets in complex diseases.