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To define the cell populations that drive joint inflammation in rheumatoid arthritis (RA), we applied single-cell RNA sequencing (scRNA-seq), mass cytometry, bulk RNA sequencing (RNA-seq) and flow cytometry to T cells, B cells, monocytes, and fibroblasts from 51 samples of synovial tissue from patients with RA or osteoarthritis (OA). Utilizing an integrated strategy based on canonical correlation analysis of 5,265 scRNA-seq profiles, we identified 18 unique cell populations. Combining mass cytometry and transcriptomics revealed cell states expanded in RA synovia: THY1(CD90)+HLA-DRAhi sublining fibroblasts, IL1B+ pro-inflammatory monocytes, ITGAX+TBX21+ autoimmune-associated B cells and PDCD1+ peripheral helper T (TPH) cells and follicular helper T (TFH) cells. We defined distinct subsets of CD8+ T cells characterized by GZMK+, GZMB+, and GNLY+ phenotypes. We mapped inflammatory mediators to their source cell populations; for example, we attributed IL6 expression to THY1+HLA-DRAhi fibroblasts and IL1B production to pro-inflammatory monocytes. These populations are potentially key mediators of RA pathogenesis.

BackgroundDetailed 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 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.MethodsMultiple 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.ResultsUpon 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 100 μg/ml of Liberase™ TL enzyme preparation. 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 diverse fibroblast phenotypes, distinct populations of memory B cells and antibody-secreting cells, and multiple CD4+ and CD8+ T-cell activation states. Bulk RNA-seq 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.ConclusionsWe 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.

Rheumatoid arthritis (RA) is a complex immune-mediated inflammatory disorder in which patients suffer from inflammatory-erosive arthritis. Recent advances on histopathology heterogeneity of RA synovial tissue revealed three distinct phenotypes based on cellular composition (pauci-immune, diffuse and lymphoid), suggesting that distinct etiologies warrant specific targeted therapy which motivates a need for cost effective phenotyping tools in preclinical and clinical settings. To this end, we developed an automated multi-scale computational pathotyping (AMSCP) pipeline for both human and mouse synovial tissue with two distinct components that can be leveraged together or independently: (1) segmentation of different tissue types to characterize tissue-level changes, and (2) cell type classification within each tissue compartment that assesses change across disease states. Here, we demonstrate the efficacy, efficiency, and robustness of the AMSCP pipeline as well as the ability to discover novel phenotypes. Taken together, we find AMSCP to be a valuable cost-effective method for both pre-clinical and clinical research. Automated pathotyping of synovial tissue in arthritis is a major unmet need. Here, the authors develop and demonstrate the efficacy of an automated tissue and cellular pathotyping tool for inflammatory arthritis. [ABSTRACT FROM AUTHOR]

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. Activated B cells and T cells accumulate within joints of patients with rheumatoid arthritis. Here, the authors use single-cell transcriptome and repertoire profiling to identify clonally expanded synovial B cells and T cells and define their phenotypes and predicted cell-cell interactions. [ABSTRACT FROM AUTHOR]

Synovial tissue inflammation is a hallmark of rheumatoid arthritis (RA). Recent work has identified prominent pathogenic cell states in inflamed RA synovial tissue, such as T peripheral helper cells; however, the epigenetic regulation of these states has yet to be defined. Here, we examine genome-wide open chromatin at single-cell resolution in 30 synovial tissue samples, including 12 samples with transcriptional data in multimodal experiments. We identify 24 chromatin classes and predict their associated transcription factors, including a CD8 + GZMK+ class associated with EOMES and a lining fibroblast class associated with AP-1. By integrating with an RA tissue transcriptional atlas, we propose that these chromatin classes represent 'superstates' corresponding to multiple transcriptional cell states. Finally, we demonstrate the utility of this RA tissue chromatin atlas through the associations between disease phenotypes and chromatin class abundance, as well as the nomination of classes mediating the effects of putatively causal RA genetic variants. The epigenetic changes underlying the heterogeneity of RA disease presentation have been the subject of intense scrutiny. In this study, the authors use multiple single-cell sequencing datasets to define 'chromatin superstates' in patients with RA, which associate with distinct transcription factors and disease phenotypes. [ABSTRACT FROM AUTHOR]

Cellular senescence plays an important role in human diseases, including osteoporosis and osteoarthritis. Senescent cells (SCs) produce the senescence-associated secretory phenotype to affect the function of neighboring cells and SCs themselves. Delayed fracture healing is common in the elderly and is accompanied by reduced mesenchymal progenitor cells (MPCs). However, the contribution of cellular senescence to fracture healing in the aged has not to our knowledge been studied. Here, we used C57BL/6J 4-month-old young and 20-month-old aged mice and demonstrated a rapid increase in SCs in the fracture callus of aged mice. The senolytic drugs dasatinib plus quercetin enhanced fracture healing in aged mice. Aged callus SCs inhibited the growth and proliferation of callus-derived MPCs (CaMPCs) and expressed high levels of TGF-[beta]1. TGF-[beta]neutralizing Ab prevented the inhibitory effects of aged callus SCs on CaMPCs and promoted fracture healing in aged mice, which was associated with increased CaMPCs and proliferating cells. Thus, fracture triggered a significant cellular senescence in the callus cells of aged mice, which inhibited MPCs by expressing TGF-[beta]1. Short-term administration of dasatinib plus quercetin depleted callus SCs and accelerated fracture healing in aged mice. Senolytic drugs represent a promising therapy, while TGF-[beta]1 signaling is a molecular mechanism for fractures in the elderly via SCs., Introduction More than 5 million long bone fractures occur annually in the United States, and 15% of them occur in people older than 50 years of age (1). Furthermore, most [...]

NOTCH-dependent signaling pathways are critical for normal bone remodeling; however, it is unclear if dysfunctional NOTCH activation contributes to inflammation-mediated bone loss, as observed in rheumatoid arthritis (RA) patients. We performed RNA sequencing and pathway analyses in mesenchymal stem cells (MSCs) isolated from transgenic TAF-expressing mice, a model of RA, to identify pathways responsible for decreased osteoblast differentiation. 53 pathways were dysregulated in MSCs from RA mice, among which expression of genes encoding NOTCH pathway members and members of the noncanonical NF-[kappa]B pathway were markedly elevated. Administration of NOTCH inhibitors to RA mice prevented bone loss and osteoblast inhibition, and CFU-fibroblasts from RA mice treated with NOTCH inhibitors formed more new bone in recipient mice with tibial defects. Overexpression of the noncanonical NF- [kappa]B subunit p52 and RELB in a murine pluripotent stem cell line increased NOTCH intracellular domain-dependent (NICD-dependent) activation of an RBPj[kappa] reporter and levels of the transcription factor HES1. TNF promoted p52/RELB binding to NICD, which enhanced binding at the RBPj[kappa] site within the Hes1 promoter. Furthermore, MSC-enriched cells from RA patients exhibited elevated levels of HES1, p52, and RELB. Together, these data indicate that persistent NOTCH activation in MSCs contributes to decreased osteoblast differentiation associated with RA and suggest that NOTCH inhibitors could prevent inflammation-mediated bone loss., Introduction Patients with chronic inflammatory diseases, such as rheumatoid arthritis (RA), often have severe systemic bone loss and increased risk of fracture due to increased bone resorption and decreased bone [...]

The cytokines RANKL and TNF activate NF-κB signaling in osteoclast precursors (OCPs) to induce osteoclast (OC) formation. Conversely, TNF can limit OC formation through NF-κB p100, which acts as an inhibitor, and TNF receptor-associated receptor 3 (TRAF3); however, a role for TRAF3 in RANKL-mediated OC formation is unknown. We found that TRAF3 limits RANKL-induced osteoclastogenesis by suppressing canonical and noncanonical NF-κB signaling. Conditional OC-specific Traf3-KO (cKO) mice had mild osteoporosis and increased OC formation. RANKL induced TRAF3 degradation via the lysosome/autophagy system. The autophagy/lysosome inhibitor chloroquine reduced RANKL-induced OC formation and function by increasing TRAF3 expression in OCPs in vitro and in vivo. Although chloroquine had no effect on basal bone resorption, it inhibited parathyroid hormone- and ovariectomy-induced OC activation in WT, but not cKO, mice. Deletion of the transcription factor gene Relb resulted in increased TRAF3 expression in OCPs, which was associated with decreased RANKL-induced TRAF3 degradation. RelB directly increased expression of BECN1, a key autophagy regulator, by binding to its promoter. These data indicate that autophagic/lysosomal degradation of TRAF3 is an important step in RANKL-induced NF-κB activation in OCPs. Furthermore, treatments that increase TRAF3 levels in OCPs, including pharmacological inhibition of its degradation with compounds such as chloroquine, may limit bone destruction in common bone diseases., Introduction Bone homeostasis is maintained by a delicate balance between osteoclastic bone resorption and osteoblastic bone formation. Disruption of this balance with a higher rate of bone resorption occurs in [...]

Cellular senescence plays important roles in age-related diseases, including musculoskeletal disorders. Senescent cells (SCs) exert a senescence-associated secretory phenotype (SASP) by producing SASP factors, some of which overlap with factors produced by inflammatory cells (Inf-Cs). However, the differences between SCs and Inf-Cs and how they interact with each other during fracture repair have not been well studied. Here, we analyzed single cell RNA sequencing data of aged mouse fracture callus stromal cells. We defined Inf-Cs as cells that express NF-kB Rela/Relb, SCs as cells that express the senescence genes, Cdkn1a, Cdkn2a or Cdkn2c, and inflammatory SCs (Inf-SCs) as cells that express both NFkB and senescence genes. Differentially expressed genes and pathway analyses revealed that Inf-SCs and SCs had a similar gene expression profile and upregulated pathways that are related to DNA damage/oxidation-reduction and cellular senescence, while Inf-Cs expressed different gene signatures and pathways from SCs and Inf-SCs, mainly related to inflammation. Cellchat software analysis indicated that SCs and Inf-SCs are potential ligand-producing cells that affect Inf-Cs as target cells. Cell culture experiments demonstrated that SC conditioned medium promoted inflammatory gene expression by callusderived mesenchymal progenitor cells, and Inf-Cs had reduced osteoblast differentiation capacity. In summary, we have identified three cell subclusters associated with inflammation and senescence in callus stromal cells, predicted potential effects of Inf-SCs and SCs on Inf-Cs by production of active ligands, and demonstrated that when mesenchymal progenitors acquire inflammatory phenotypes their osteogenic potential is reduced. [ABSTRACT FROM AUTHOR]

TGFβ1 induces age-related bone loss by promoting degradation of TNF receptor-associated factor 3 (TRAF3), levels of which decrease in murine and human bone during aging. We report that a subset of neutrophils (TGFβ1+CCR5+) is the major source of TGFβ1 in murine bone. Their numbers are increased in bone marrow (BM) of aged wild-type mice and adult mice with TRAF3 conditionally deleted in mesenchymal progenitor cells (MPCs), associated with increased expression in BM of the chemokine, CCL5, suggesting that TRAF3 in MPCs limits TGFβ1+CCR5+ neutrophil numbers in BM of young mice. During aging, TGFβ1-induced TRAF3 degradation in MPCs promotes NF-κB-mediated expression of CCL5 by MPCs, associated with higher TGFβ1+CCR5+ neutrophil numbers in BM where they induce bone loss. TGFβ1+CCR5+ neutrophils decreased bone mass in male mice. The FDA-approved CCR5 antagonist, maraviroc, reduced TGFβ1+CCR5+ neutrophil numbers in BM and increased bone mass in aged mice. 15-mon-old mice with TGFβRII specifically deleted in MPCs had lower numbers of TGFβ1+CCR5+ neutrophils in BM and higher bone volume than wild-type littermates. We propose that pharmacologic reduction of TGFβ1+CCR5+ neutrophil numbers in BM could treat or prevent age-related osteoporosis. Age-related osteoporosis is known to be dependent on TGFβ1 signalling. Here the authors show that a subset of neutrophils (identified by TGFβ1+CCR5+) increase during aging in mouse bone marrow resulting in bone loss, while a CCR5 antagonist can reduce the neutrophil numbers and increase bone mass in aged mice. [ABSTRACT FROM AUTHOR]

TNF and RANKL mediate bone destruction in common bone diseases, including osteoarthritis and RA. They activate NF-κB canonical signaling directly in osteoclast precursors (OCPs) to induce osteoclast formation in vitro. However, unlike RANKL, TNF does not activate the alternative NF-κB pathway efficiently to process the IκB protein NF-κB p100 to NF-κB p52, nor does it appear to induce osteoclast formation in vivo in the absence of RANKL. Here, we show that TNF limits RANKL- and TNF-induced osteoclast formation in vitro and in vivo by increasing NF-κB p100 protein accumulation in OCPs. In contrast, TNF induced robust osteoclast formation in vivo in mice lacking RANKL or RANK when the mice also lacked NF-κB p100, and TNF-Tg mice lacking NF-κB p100 had more severe joint erosion and inflammation than did TNF-Tg littermates. TNF, but not RANKL, increased OCP expression of TNF receptor-associated factor 3 (TRAF3), an adapter protein that regulates NF-κB p100 levels in B cells. TRAF3 siRNA prevented TNF-induced NF-κB p100 accumulation and inhibition of osteoclastogenesis. These findings suggest that upregulation of TRAF3 or NF-κB p100 expression or inhibition of NF-κB p100 degradation in OCPs could limit bone destruction and inflammation-induced bone loss in common bone diseases., Introduction Osteoclasts, the cells that degrade bone, play a central role in bone destruction in common erosive bone diseases, including postmenopausal osteoporosis, RA, and periodontitis (1). In these conditions there [...]

Notch signaling is a key mechanism in the control of embryogenesis. However, its in vivo function during mesenchymal cell differentiation, and, specifically, in bone homeostasis, remains largely unknown. Here, we show that osteoblast- specific gain of Notch function causes severe osteosclerosis owing to increased proliferation of immature osteoblasts. Under these pathological conditions, Notch stimulates early osteoblastic proliferation by upregulating the genes encoding cyclin D, cyclin E and Sp7 (osterix). The intracellular domain of Notchl also regulates terminal osteoblastic differentiation by directly binding Runx2 and repressing its transactivation function. In contrast, loss of all Notch signaling in osteoblasts, generated by deletion of the genes encoding presenilin-1 and presenilin-2 in bone, is associated with late-onset, age-related osteoporosis, which in turn results from increased osteoblast-dependent osteoblastic activity due to decreased osteoprotegerin mRNA expression in these cells. Together, these findings highlight the potential dimorphic effects of Notch signaling in bone homeostasis and may provide direction for novel therapeutic applications., Evolutionarily conserved Notch signaling has a crucial role in cell fate determination and various developmental processes, as it translates cell-cell interactions into specific transcriptional programs (1,2). Temporal and spatial modulation [...]

The Cartilage Association Protein (CRTAP) which copurifies with a collagen prolyl 3-hydroxylase activity is involved in osteogenesis imperfecta and mutations are associated with brittle bones in patients with a spectrum of mild to severe recessive osteogenesis imperfecta. Hence altered CRTAP function and prolyl 3-hydroxylation contribute to the connective tissue disease.

Structural allograft healing is limited because of a lack of vascularization and remodeling. To study this we developed a mouse model that recapitulates the clinical aspects of live autograft and processed allograft healing. Gene expression analyses showed that there is a substantial decrease in the genes encoding RANKL and VEGF during allograft healing. Loss-of-function studies showed that both factors are required for autograft healing. To determine whether addition of these signals could stimulate allograft vascularization and remodeling, we developed a new approach in which rAAV can be freeze-dried onto the cortical surface without losing infectivity. We show that combination rAAV-RANKL- and rAAV-VEGF-coated allografts show marked remodeling and vascularization, which leads to a new bone collar around the graft. In conclusion, we find that RANKL and VEGF are necessary and sufficient for efficient autograft remodeling and can be transferred using rAAV to revitalize structural allografts., Author(s): Hiromu Ito [1, 2]; Mette Koefoed [1, 3]; Prarop Tiyapatanaputi [1]; Kirill Gromov [1, 3]; J Jeffrey Goater [1]; Jonathan Carmouche [1]; Xinping Zhang [1]; Paul T Rubery [1]; [...]