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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 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 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. Conclusions 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. Electronic supplementary material The online version of this article (10.1186/s13075-018-1631-y) contains supplementary material, which is available to authorized users.

Introduction The c-Jun N-terminal kinase (JNK) is a key regulator of matrix metalloproteinase (MMP) and cytokine production in rheumatoid arthritis (RA) and JNK deficiency markedly protects mice in animal models of arthritis. Cytokine-induced JNK activation is strictly dependent on the mitogen-activated protein kinase kinase 7 (MKK7) in fibroblast-like synoviocytes (FLS). Therefore, we evaluated whether targeting MKK7 using anti-sense oligonucleotides (ASO) would decrease JNK activation and severity in K/BxN serum transfer arthritis. Methods Three 2'-O-methoxyethyl chimeric ASOs for MKK7 and control ASO were injected intravenously in normal C57BL/6 mice. PBS, control ASO or MKK7 ASO was injected from Day -8 to Day 10 in the passive K/BxN model. Ankle histology was evaluated using a semi-quantitative scoring system. Expression of MKK7 and JNK pathways was evaluated by quantitative PCR and Western blot analysis. Results MKK7 ASO decreased MKK7 mRNA and protein levels in ankles by about 40% in normal mice within three days. There was no effect of control ASO on MKK7 expression and MKK7 ASO did not affect MKK3, MKK4 or MKK6. Mice injected with MKK7 ASO had significantly less severe arthritis compared with control ASO (P < 0.01). Histologic evidence of synovial inflammation, bone erosion and cartilage damage was reduced in MKK7 ASO-treated mice (P < 0.01). MKK7 deficiency decreased phospho-JNK and phospho-c-Jun in ankle extracts (P < 0.05), but not phospho-MKK4. Interleukin-1beta (IL-1β), MMP3 and MMP13 gene expression in ankle joints were decreased by MKK7 ASO (P < 0.01). Conclusions MKK7 plays a critical regulatory role in the JNK pathway in a murine model of arthritis. Targeting MKK7 rather than JNK could provide site and event specificity when treating synovitis.

PUMA (p53-upregulated modulator of apoptosis) is a pro-apoptotic gene that can induce rapid cell death through a p53-dependent mechanism. However, the efficacy of PUMA gene therapy to induce synovial apoptosis in rheumatoid arthritis might have limited efficacy if p53 expression or function is deficient. To evaluate this issue, studies were performed to determine whether p53 is required for PUMA-mediated apoptosis in fibroblast-like synoviocytes (FLS). p53 protein was depleted or inhibited in human FLS by using p53 siRNA or a dominant-negative p53 protein. Wild-type and p53-/- murine FLS were also examined to evaluate whether p53 is required. p53-deficient or control FLS were transfected with PUMA cDNA or empty vector. p53 and p21 expression were then determined by Western blot analysis. Apoptosis was assayed by ELISA to measure histone release and caspase-3 activation, or by trypan blue dye exclusion to measure cell viability. Initial studies showed that p53 siRNA decreased p53 expression by more than 98% in human FLS. Loss of p53 increased the growth rate of cells and suppressed p21 expression. However, PUMA still induced apoptosis in control and p53-deficient FLS after PUMA cDNA transfection. Similar results were observed in p53-/- murine FLS or in human FLS transfected with a dominant-negative mutant p53 gene. These data suggest that PUMA-induced apoptosis in FLS does not require p53. Therefore, approaches to gene therapy that involve increasing PUMA expression could be an effective inducer of synoviocyte cell death in rheumatoid arthritis regardless of the p53 status in the synovium.

The objective of this study was to evaluate the use of real-time quantitative PCR (Q-PCR) using a cellular standard to detect changes in gene expression in synovial tissue samples that could potentially serve as such biomarkers correlating with clinical disease activity. Therefore, the effects of treatment with corticosteroids, a known effective therapy, were analyzed in patients with active rheumatoid arthritis. Patients were randomized to receive either oral prednisolone (n = 10, 60 mg daily for the first and 40 mg daily for the second week) or placebo (n = 11). All patients underwent an arthroscopic synovial biopsy procedure directly before and after 14 days of treatment. Real-time Q-PCR was used to quantify gene expression of tumour necrosis factor alpha, IL-1β, IL-6 and MMP-1 in the synovial tissue samples. The values were expressed as relative units compared with a cellular-based standard. Statistical analysis was performed using an analysis of covariance model. The mean DAS28 was 2.0 units lower (95% confidence interval, 1.0–3.0) after prednisolone therapy compared with placebo. The mean DAS28 (± standard deviation) decreased (from 6.27 ± 0.95 to 4.11 ± 1.43) after prednisolone therapy, but not in the placebo group. For gene expression of IL-8 and MMP-1, the estimated effect of prednisolone compared with placebo was large, and the confidence intervals excluded the likelihood of no effect. A clear trend towards reduction was seen in IL-β and tumour necrosis factor alpha mRNA expression in the prednisolone group, but confidence intervals included the value for no effect. The results of this study show that mRNA expression of IL-8 and MMP-1 quantified by Q-PCR may serve as biomarkers in small proof of principle trials designed to screen for potential efficacy in rheumatoid arthritis patients.

Studies of cytokine expression in rheumatoid arthritis have provided key insights into the pathogenesis of disease and have offered clues for effective therapy. Patterns of T-cell products in chronic rheumatoid synovitis suggest that T helper type 1 cells contribute to the perpetuation of disease. However, there is no guarantee that the mechanisms of late disease are identical to very early rheumatoid arthritis. Evaluation of the cytokine profile at the earliest time points after onset of symptoms could identify novel targets that prevent progression to chronic arthritis.

Rheumatoid arthritis (RA) is an immune-mediated disease with synovial inflammation and invasion of the extracellular matrix. While adaptive immunity plays a key role in the pathogenesis of RA, the contribution of innate immune responses has been increasingly appreciated in recent years. Toll-like receptors (TLRs) and cytokines in the rheumatoid synovium can activate signal transduction pathways that induce proinflammatory genes and perpetuate synovitis. Understanding these signaling mechanisms helps us understand the pathogenesis of disease and identify novel therapeutic targets. Among the signaling pathways that are potentially involved in inflammation, the IKK-related kinase, inducible IKK (IKKi or IKKe), appears to play a key role in innate immune responses in the joint and modulates synovial inflammatory responses. This kinase, along with a second IKK-related kinase TANK binding kinase 1 (TBK1), was originally identified as a NF-κB activating enzyme that phosphorylates IkB. It is now clear, however, that this represents only one of several substrates for this family. For instance, IKKi also phosphorylates interferon regulatory factor (IRF) and coordinates the activation of IRF and NF-κB after TLR ligation. IKKi may also link the NF-κB and CCAAT enhancer binding protein (C/EBP) pathways in lipopolysaccharide-stimulated cells. The novel roles for IKKi suggests that it contributes to the signaling pathways involved in synovial inflammation. Moreover, IKKi appears to play a key role in the establishment of an 'antiviral state' through the activation of IRF3 and c-Jun with subsequent production of interferon beta (IFN-β) and other genes involved in host defense. We previously demonstrated that IKKi is constitutively expressed in RA synovial tissue and fibroblast-like synoviocytes (FLS), and that the gene can be induced by cytokine stimulation. More recent studies now show that functional activity of IKKi in cultured synoviocytes is rapidly increased by exposure to proinflammatory cytokines and TLR agonists. We have also shown that IRF3 is activated in RA synovium, which represents an early step in the IKKi-mediated expression of antiviral genes like IFN-β. Additional preliminary data with human FLS suggest that the IKKi pathway can activate other signaling cascades, such as c-Jun, which play a role in assembly of the interferon enhanceosome to increase IFN-β expression. Using FLS from IKKi knockout mice, we have shown that IKKi can act as a key link between TLR/cytokine receptor ligation and expression of cytokines and matrix metalloproteinases. IKKi might serve in an alternative signaling pathway for activation of innate immunity in RA and establishing an anti-viral state in the synovium, especially through the activation of IRF3-driven and c-Jun-driven genes.

The mitogen activated protein (MAP) kinases represent another attractive target for rheumatoid arthritis (RA) because they can regulate cell proliferation, apoptosis, cytokine expression, and metalloproteinase production. They represent a complex, interrelated signal transduction mechanism that integrates extracellular stresses and induces an appropriate cellular response. The three major MAP kinase families, c-Jun-N-terminal kinase (JNK), extracellular regulating kinase (ERK) and p38 kinase, differ in their substrate specificity and subsequent responses to stress depending on the cell type and the environmental influences. The MAP kinases regulate various genes via both transcriptional and post-transcriptional mechanisms. The upstream MAP kinase kinases (MAPKK) serve as regulators of MAP kinase activity by phosphorylating specific threonine and tyrosine residues. MAPKKs are, in turn, regulated, by MAPKK kinases (MAPKKK or MAP3K). The p38 MAP kinase is of particular interest and several inhibitors have progressed into clinical trials. There are at least four isoforms of p38 (α, β, γ and δ), although the α form is probably the most important in macrophages for cytokine regulation. Many of the cytokine regulatory effects appear to be mediated through the downstream p38 substrate MAPKAP-2. Some evidence suggests that tumor necrosis factor production, in particular, is regulated by this kinase. Preclinical models demonstrate that p38 inhibitors are effective in a number of animal models of arthritis, including murine collagen-induced arthritis. Of note, p38 inhibitors also suppress joint destruction in these models, perhaps due to a combination of indirect effects on cytokine expression and a direct effect on metalloproteinase production. In vitro studies previously identified the MAPKKs MKK3 and MKK6 as the primary regulators of p38 phosphorylation and activation. To investigate a potential role for MKK3 and MKK6 in RA, we evaluated their expression and regulation in RA synovium and cultured fibroblast-like synoviocytes (FLS). Immunohistochemistry demonstrated that MKK3 and MKK6 are expressed in RA and osteoarthritis (OA) synovium. Digital image analysis showed no significant differences between OA and RA with regard to expression or distribution. However, phosphorylated MKK3/MKK6 expression was significantly higher in RA synovium and was localized to the sublining mononuclear cells and the intimal lining. Western blot analysis of synovial tissue lysates confirm the increased expression of phosphorylated MKK3/MKK6 in RA. Western blot analysis demonstrated constitutive expression of MKK3 and MKK6 in RA and OA FLS. Phospho-MKK3 levels were low in medium-treated FLS, but were rapidly increased by IL-1 and tumor necrosis factor alpha, although phospho-MKK6 levels only modestly increased. p38 co-immunoprecipitated with MKK3 and MKK6 from cytokine-stimulated FLS, and the complex phosphorylated ATF2 in an in vitro kinase assay. Studies using dominant negative constructs in cultured synoviocytes suggest that both are required for full activation of p38. These data are the first documentation of MKK3 and MKK6 activation in human inflammatory disease. By forming a complex with p38 in synovial tissue and FLS, these kinases can potentially be targeted to regulate the production of proinflammatory cytokine production in inflamed synovium.

The molecular pathogenesis of rheumatoid arthritis (RA) is still poorly understood and its clinical course can vary widely. In a recent report we noted heterogeneity in global gene expression signatures between synovial tissue specimens from different patients with RA. The results of the present study support the view that clinically diagnosed RA is a heterogeneous disease that is featured not only at the whole synovial tissue level but also at the level of FLS cultured from those tissues. One of the most impressive features of our 24k cDNA microarray profiling studies is the clear correlation of the FLS phenotype with that of paired synovial tissue from which the cells were derived. One class of FLS is tightly related to the presence of lymphocytes in the lesions whereas the other class of FLS suggests that synoviocyte-mediated invasion appears to be less dependent on infiltrating immune cells. Clearly, the list of genes that are differentially expressed between the FLS subgroups facilitates our understanding of the pathophysiology of the distinct groups of rheumatoid FLS. These data reveal features of fibrosis as the hallmark of FLS derived from a high inflammatory lesion, whereas FLS that are characterized by deregulated growth appear to constitute a characteristic feature of low inflammatory tissues. These data support the notion that heterogeneity between synovial tissues is reflected in the FLS as a stable trait and provide a molecular basis for the well recognized but as yet poorly understood heterogeneity in RA.

Synovial biomarker analysis in rheumatoid arthritis can be used to evaluate drug effect in clinical trials of novel therapeutic agents. Previous studies of synovial gene expression for these studies have mainly relied on histological methods including immunohistochemistry and in situ hybridization. To increase the reliability of mRNA measurements on small synovial tissue samples, we developed and validated real time quantitative PCR (Q-PCR) methods on biopsy specimens. RNA was isolated from synovial tissue and cDNA was prepared. Cell-based standards were prepared from mitogen-stimulated peripheral blood mononuclear cells. Real time PCR was performed using TaqMan chemistry to quantify gene expression relative to the cell-based standard. Application of the cellular standard curve method markedly reduced intra- and inter-assay variability and corrected amplification efficiency errors compared with the C(t) method. The inter-assay coefficient of variation was less than 25% over time. Q-PCR methods were validated by demonstrating increased expression of IL-1β and IL-6 expression in rheumatoid arthritis synovial samples compared with osteoarthritis synovium. Based on determinations of sampling error and coefficient of variation, twofold differences in gene expression in serial biopsies can be detected by assaying approximately six synovial tissue biopsies from 8 to 10 patients. These data indicate that Q-PCR is a reliable method for determining relative gene expression in small synovial tissue specimens. The technique can potentially be used in serial biopsy studies to provide insights into mechanism of action and therapeutic effect of new anti-inflammatory agents.