Gary S. Firestein

Rheumatoid arthritis (RA) is a systemic autoimmune disease currently with no universally highly effective prevention strategies. Identifying pathogenic immune phenotypes in at-risk populations prior to clinical onset is crucial to establishing effective prevention strategies. Here, we applied multimodal single-cell technologies (mass cytometry and CITE-Seq) to characterize the immunophenotypes in blood from at-risk individuals (ARIs) identified through the presence of serum antibodies against citrullinated protein antigens (ACPAs) and/or first-degree relative (FDR) status, as compared with patients with established RA and people in a healthy control group. We identified significant cell expansions in ARIs compared with controls, including CCR2 ⁺ CD4 ⁺ T cells, T peripheral helper (Tph) cells, type 1 T helper cells, and CXCR5 ⁺ CD8 ⁺ T cells. We also found that CD15 ⁺ classical monocytes were specifically expanded in ACPA-negative FDRs, and an activated PAX5 ^lo naive B cell population was expanded in ACPA-positive FDRs. Further, we uncovered the molecular phenotype of the CCR2 ⁺ CD4 ⁺ T cells, expressing high levels of Th17- and Th22-related signature transcripts including CCR6 , IL23R , KLRB1 , CD96 , and IL22 . Our integrated study provides a promising approach to identify targets to improve prevention strategy development for RA.

The mechanisms responsible for the distribution and severity of joint involvement in rheumatoid arthritis (RA) are not known. To explore whether site-specific fibroblast-like synoviocyte (FLS) biology might be associated with location-specific synovitis and explain the predilection for hand (wrist/metacarpal phalangeal joints) involvement in RA, we generated transcriptomic and chromatin accessibility data from FLS to identify the transcription factors and pathways. Networks were constructed by integration of chromatin accessibility and gene expression data. Analysis revealed joint-specific patterns of FLS phenotype, with proliferative, migratory, proinflammatory, and matrix-degrading characteristics observed in resting FLS derived from the hand joints compared with hip or knee. TNF stimulation amplified these differences, with greater enrichment of proinflammatory and proliferative genes in hand FLS compared with hip and knee FLS. Hand FLS also had the greatest expression of markers associated with an “activated” state relative to the “resting” state, with the greatest cytokine and MMP expression in TNF-stimulated hand FLS. Predicted differences in proliferation and migration were biologically validated with hand FLS exhibiting greater migration and cell growth than hip or knee FLS. Distinctive joint-specific FLS biology associated with a more aggressive inflammatory response might contribute to the distribution and severity of joint involvement in RA.

The cytokine profile during acute chikungunya infection that predicts future chronic arthritis has not yet been investigated. We conducted a nested case-control study comparing serum cytokine concentrations during acute chikungunya infection in cases (n = 121) that reported the presence of chronic joint pain versus age- and gender-matched controls (n = 121) who reported recovery at 20 months post infection. We observed that a robust cytokine response during acute infection was correlated with a decreased incidence of chronic joint pain and that low TNFα, IL-13, IL-2, and IL-4 during acute infection was predictive of chronic joint pain. These data suggest that a robust cytokine response is necessary for viral clearance and cytokines that are related to immune tolerance during acute infection may be protective for chronic arthritis pathogenesis.

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 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. 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. 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 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. 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.

To define the cell populations in rheumatoid arthritis (RA) driving joint inflammation, we applied single-cell RNA-seq (scRNA-seq), mass cytometry, bulk RNA-seq, and flow cytometry to sorted T cells, B cells, monocytes, and fibroblasts from 51 synovial tissue RA and osteoarthritis (OA) patient samples. Utilizing an integrated computational strategy based on canonical correlation analysis to 5,452 scRNA-seq profiles, we identified 18 unique cell populations. Combining mass cytometry and transcriptomics together revealed cell states expanded in RA synovia: THY1 ⁺ HLA ^high sublining fibroblasts (OR=33.8), IL1B ⁺ pro-inflammatory monocytes (OR=7.8), CD11c ⁺ T-bet ⁺ autoimmune-associated B cells (OR=5.7), and PD-1 ⁺ Tph/Tfh (OR=3.0). We also defined CD8 ⁺ T cell subsets characterized by GZMK ⁺ , GZMB ⁺ , and GNLY ⁺ expression. Using bulk and single-cell data, we mapped inflammatory mediators to source cell populations, for example attributing IL6 production to THY1 ⁺ HLA ^high fibroblasts and naïve B cells, and IL1B to pro-inflammatory monocytes. These populations are potentially key mediators of RA pathogenesis.

Epigenetics contributes to the pathogenesis of immune-mediated diseases like rheumatoid arthritis (RA). Here we show the first comprehensive epigenomic characterization of RA fibroblast-like synoviocytes (FLS), including histone modifications (H3K27ac, H3K4me1, H3K4me3, H3K36me3, H3K27me3, and H3K9me3), open chromatin, RNA expression and whole-genome DNA methylation. To address complex multidimensional relationship and reveal epigenetic regulation of RA, we perform integrative analyses using a novel unbiased method to identify genomic regions with similar profiles. Epigenomically similar regions exist in RA cells and are associated with active enhancers and promoters and specific transcription factor binding motifs. Differentially marked genes are enriched for immunological and unexpected pathways, with “Huntington’s Disease Signaling” identified as particularly prominent. We validate the relevance of this pathway to RA by showing that Huntingtin-interacting protein-1 regulates FLS invasion into matrix. This work establishes a high-resolution epigenomic landscape of RA and demonstrates the potential for integrative analyses to identify unanticipated therapeutic targets.

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.

Rheumatoid arthritis is a synovial inflammatory disease marked by joint infiltration by immune cells and damage to the extracellular matrix. Although genetics plays a critical role in heritability and its pathogenesis, the relative lack of disease concordance in identical twins suggests that noncoding influences can affect risk and severity. Environmental stress, which can be reflected in the genome as altered epigenetic marks, also contributes to gene regulation and contributes to disease mechanisms. Studies on DNA methylation suggest that synovial cells, most notably fibroblast-like synoviocytes, are imprinted in rheumatoid arthritis with epigenetic marks and subsequently assume an aggressive phenotype. Even more interesting, the synoviocyte marks are not only disease specific but can vary depending on the joint of origin. Understanding the epigenetic landscape using unbiased methods can potentially identify nonobvious pathways and genes that that are responsible for synovial inflammation as well as the diversity of responses to targeted agents. The information can also be leveraged to identify novel therapeutic approaches.

Rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) display unique aggressive behavior, invading the articular cartilage and promoting inflammation. Using an integrative analysis of RA risk alleles, the transcriptome and methylome in RA FLS, we recently identified the limb bud and heart development (LBH) gene as a key dysregulated gene in RA and other autoimmune diseases. Although some evidence suggests that LBH could modulate the cell cycle, the precise mechanism is unknown and its impact on inflammation in vivo has not been defined. Our cell cycle analysis studies show that LBH deficiency in FLS leads to S-phase arrest and failure to progress through the cell cycle. LBH-deficient FLS had increased DNA damage and reduced expression of the catalytic subunit of DNA polymerase α. Decreased DNA polymerase α was followed by checkpoint arrest due to phosphorylation of checkpoint kinase 1. Because DNA fragments can increase arthritis severity in preclinical models, we then explored the effect of LBH deficiency in the K/BxN serum transfer model. Lbh knockout exacerbated disease severity, which is associated with elevated levels of IL-1β and checkpoint kinase 1 phosphorylation. These studies indicate that LBH deficiency induces S-phase arrest that, in turn, exacerbates inflammation. Because LBH gene variants are associated with type I diabetes mellitus, systemic lupus erythematosus, RA, and celiac disease, these results suggest a general mechanism that could contribute to immune-mediated diseases.