Droplet-based single-cell RNA-seq has emerged as a powerful technique for massively parallel cellular profiling. While this approach offers the exciting promise to deconvolute cellular heterogeneity ...in diseased tissues, the lack of cost-effective and user-friendly instrumentation has hindered widespread adoption of droplet microfluidic techniques. To address this, we developed a 3D-printed, low-cost droplet microfluidic control instrument and deploy it in a clinical environment to perform single-cell transcriptome profiling of disaggregated synovial tissue from five rheumatoid arthritis patients. We sequence 20,387 single cells revealing 13 transcriptomically distinct clusters. These encompass an unsupervised draft atlas of the autoimmune infiltrate that contribute to disease biology. Additionally, we identify previously uncharacterized fibroblast subpopulations and discern their spatial location within the synovium. We envision that this instrument will have broad utility in both research and clinical settings, enabling low-cost and routine application of microfluidic techniques.
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.
Autoimmune forms of inflammatory arthritis, such as Rheumatoid Arthritis (RA), are clinically heterogeneous in presentation and disease course. Treatment-related outcomes vary despite patient ...exposure to similar treatment strategies. It is likely that variation seen in synovial pathogenesis influences outcomes and is heterogeneous outcomes influenced by patient factors, including environmental exposures, microbiota, behaviors, timely access to therapy, and synovial cell variation. Patients’ unique complex factors manifest as specific synovial phenotypes characterized by clusters of synovial cell types and states. Precision medicine aims to use such clinical and biological data to identify the right treatment for the right patient at the right time, enabling patients to achieve sustained remission. Identifying synovial targets susceptible to a given treatment, enabling the choice of effective therapy for a given patient, will realize the goals of precision medicine. Over the last 7 years, improved acquisition and processing of synovial tissue obtained by ultra-sound guided biopsy has enabled researchers to define synovial pathotypes using histologic features and predominant cell types associated with clinical manifestations. Technical advances have enabled single-cell simultaneous sequencing of proteins and gene expression that, through increasingly sophisticated bioinformatics methods, have taken transcriptional and proteomic data to identify diverse and novel cell types and states that cluster in the RA synovium to further define patient subgroups. Synovial pathotypes and endotypes are now integrated into clinical studies and trials to explain clinical heterogeneity in disease course and treatment response. Rapidly evolving clinical-translational research has linked an expanded understanding of RA synovial pathogenesis with clinically meaningful subgroups and treatment outcomes and the clinical heterogeneity in RA.
Macrophages tailor their function according to the signals found in tissue microenvironments, assuming a wide spectrum of phenotypes. A detailed understanding of macrophage phenotypes in human ...tissues is limited. Using single-cell RNA sequencing, we defined distinct macrophage subsets in the joints of patients with the autoimmune disease rheumatoid arthritis (RA), which affects ~1% of the population. The subset we refer to as HBEGF
inflammatory macrophages is enriched in RA tissues and is shaped by resident fibroblasts and the cytokine tumor necrosis factor (TNF). These macrophages promoted fibroblast invasiveness in an epidermal growth factor receptor-dependent manner, indicating that intercellular cross-talk in this inflamed setting reshapes both cell types and contributes to fibroblast-mediated joint destruction. In an ex vivo synovial tissue assay, most medications used to treat RA patients targeted HBEGF
inflammatory macrophages; however, in some cases, medication redirected them into a state that is not expected to resolve inflammation. These data highlight how advances in our understanding of chronically inflamed human tissues and the effects of medications therein can be achieved by studies on local macrophage phenotypes and intercellular interactions.
Medication adherence in patients with rheumatoid arthritis (RA) is typically suboptimal. Nonadherence has been associated with symptom worsening and increased disability. We systematically reviewed ...published clinical studies to evaluate methotrexate (MTX) adherence and persistence, factors associated with MTX adherence and persistence, and the effect of MTX nonadherence on clinical outcomes in RA.
MEDLINE and Embase were systematically searched (inception to February 2016) using relevant keywords. Observational or interventional clinical studies in patients with RA that specifically reported adherence to or persistence with MTX were included. Data were extracted using a predesigned, standardized template that included study design, patient demographics, and relevant outcomes. Main outcomes were MTX adherence and persistence rates in patients with RA treated with MTX and factors associated with MTX adherence and persistence.
Of 365 references screened, 31 articles met inclusion criteria and another 10 were identified from searching reference lists. Estimates of MTX adherence varied from study to study because of heterogeneity in patient populations, duration of followup, definitions of adherence, and methods of assessment. Rates of MTX persistence ranged from 50% to 94% at 1 year and 25% to 79% at 5 years. No clear trends were identified in factors that influence MTX adherence and persistence. Two studies suggested that MTX adherence was associated with superior clinical outcomes.
MTX adherence and persistence are highly variable in patients with RA. Research is necessary to determine the effect of nonadherence on health outcomes and to identify independent predictors of nonadherence to inform evidence-based interventions.
"Legacy" patient-reported outcome measures (PROs) have been used for decades; however, they have many limitations. The National Institutes of Health-funded PRO Measurement Information System (PROMIS) ...was developed to be a generic, flexible, precise, and reliable tool to measure core and additional domains of physical and emotional health and social well-being. Unlike Legacy PROs, PROMIS measures can be implemented across diseases, and use a common T-score metric-based scoring system derived using item response theory. PROMIS measure scores have potential to predict scores of Legacy PROs and could be the only set of measures needed to assess PROs.
To evaluate clinical remission with subcutaneous abatacept plus methotrexate (MTX) and abatacept monotherapy at 12 months in patients with early rheumatoid arthritis (RA), and maintenance of ...remission following the rapid withdrawal of all RA treatment.
In the Assessing Very Early Rheumatoid arthritis Treatment phase 3b trial, patients with early active RA were randomised to double-blind, weekly, subcutaneous abatacept 125 mg plus MTX, abatacept 125 mg monotherapy, or MTX for 12 months. Patients with low disease activity (Disease Activity Score (DAS)28 (C reactive protein (CRP)) <3.2) at month 12 entered a 12-month period of withdrawal of all RA therapy. The coprimary endpoints were the proportion of patients with DAS28 (CRP) <2.6 at month 12 and both months 12 and 18, for abatacept plus MTX versus MTX.
Patients had <2 years of RA symptoms, DAS28 (CRP) ≥3.2, anticitrullinated peptide-2 antibody positivity and 95.2% were rheumatoid factor positive. For abatacept plus MTX versus MTX, DAS28 (CRP) <2.6 was achieved in 60.9% versus 45.2% (p=0.010) at 12 months, and following treatment withdrawal, in 14.8% versus 7.8% (p=0.045) at both 12 and 18 months. DAS28 (CRP) <2.6 was achieved for abatacept monotherapy in 42.5% (month 12) and 12.4% (both months 12 and 18). Both abatacept arms had a safety profile comparable with MTX alone.
Abatacept plus MTX demonstrated robust efficacy compared with MTX alone in early RA, with a good safety profile. The achievement of sustained remission following withdrawal of all RA therapy suggests an effect of abatacept's mechanism on autoimmune processes.
NCT01142726.
Objective
In this study, we sought to refine histologic scoring of rheumatoid arthritis (RA) synovial tissue by training with gene expression data and machine learning.
Methods
Twenty histologic ...features were assessed in 129 synovial tissue samples (n = 123 RA patients and n = 6 osteoarthritis OA patients). Consensus clustering was performed on gene expression data from a subset of 45 synovial samples. Support vector machine learning was used to predict gene expression subtypes, using histologic data as the input. Corresponding clinical data were compared across subtypes.
Results
Consensus clustering of gene expression data revealed 3 distinct synovial subtypes, including a high inflammatory subtype characterized by extensive infiltration of leukocytes, a low inflammatory subtype characterized by enrichment in pathways including transforming growth factor β, glycoproteins, and neuronal genes, and a mixed subtype. Machine learning applied to histologic features, with gene expression subtypes serving as labels, generated an algorithm for the scoring of histologic features. Patients with the high inflammatory synovial subtype exhibited higher levels of markers of systemic inflammation and autoantibodies. C‐reactive protein (CRP) levels were significantly correlated with the severity of pain in the high inflammatory subgroup but not in the others.
Conclusion
Gene expression analysis of RA and OA synovial tissue revealed 3 distinct synovial subtypes. These labels were used to generate a histologic scoring algorithm in which the histologic scores were found to be associated with parameters of systemic inflammation, including the erythrocyte sedimentation rate, CRP level, and autoantibody levels. Comparison of gene expression patterns to clinical features revealed a potentially clinically important distinction: mechanisms of pain may differ in patients with different synovial subtypes.
CD4
T cells are central mediators of autoimmune pathology; however, defining their key effector functions in specific autoimmune diseases remains challenging. Pathogenic CD4
T cells within affected ...tissues may be identified by expression of markers of recent activation. Here we use mass cytometry to analyse activated T cells in joint tissue from patients with rheumatoid arthritis, a chronic immune-mediated arthritis that affects up to 1% of the population. This approach revealed a markedly expanded population of PD-1
CXCR5
CD4
T cells in synovium of patients with rheumatoid arthritis. However, these cells are not exhausted, despite high PD-1 expression. Rather, using multidimensional cytometry, transcriptomics, and functional assays, we define a population of PD-1
CXCR5
'peripheral helper' T (T
) cells that express factors enabling B-cell help, including IL-21, CXCL13, ICOS, and MAF. Like PD-1
CXCR5
T follicular helper cells, T
cells induce plasma cell differentiation in vitro through IL-21 secretion and SLAMF5 interaction (refs 3, 4). However, global transcriptomics highlight differences between T
cells and T follicular helper cells, including altered expression of BCL6 and BLIMP1 and unique expression of chemokine receptors that direct migration to inflamed sites, such as CCR2, CX3CR1, and CCR5, in T
cells. T
cells appear to be uniquely poised to promote B-cell responses and antibody production within pathologically inflamed non-lymphoid tissues.
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