Defining inflammatory cell states in rheumatoid arthritis joint synovial tissues by integrating single-cell transcriptomics and mass cytometry.

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.

The pathogenesis of rheumatoid arthritis.

Significant recent progress in understanding rheumatoid arthritis (RA) pathogenesis has led to improved treatment and quality of life. The introduction of targeted-biologic and -synthetic disease modifying anti-rheumatic drugs (DMARDs) has also transformed clinical outcomes. Despite this, RA remains a life-long disease without a cure. Unmet needs include partial response and non-response to treatment in many patients, failure to achieve immune homeostasis or drug free remission, and inability to repair damaged tissues. RA is now recognized as the end of a multi-year prodromal phase in which systemic immune dysregulation, likely beginning in mucosal surfaces, is followed by a symptomatic clinical phase. Inflammation and immune reactivity are primarily localized to the synovium leading to pain and articular damage, but is also associated with a broader series of comorbidities. Here, we review recently described immunologic mechanisms that drive breach of tolerance, chronic synovitis, and remission.

Deconstruction of rheumatoid arthritis synovium defines inflammatory subtypes.

Rheumatoid arthritis is a prototypical autoimmune disease that causes joint inflammation and destruction1. There is currently no cure for rheumatoid arthritis, and the effectiveness of treatments varies across patients, suggesting an undefined pathogenic diversity1,2. Here, to deconstruct the cell states and pathways that characterize this pathogenic heterogeneity, we profiled the full spectrum of cells in inflamed synovium from patients with rheumatoid arthritis. We used multi-modal single-cell RNA-sequencing and surface protein data coupled with histology of synovial tissue from 79 donors to build single-cell atlas of rheumatoid arthritis synovial tissue that includes more than 314,000 cells. We stratified tissues into six groups, referred to as cell-type abundance phenotypes (CTAPs), each characterized by selectively enriched cell states. These CTAPs demonstrate the diversity of synovial inflammation in rheumatoid arthritis, ranging from samples enriched for T and B cells to those largely lacking lymphocytes. Disease-relevant cell states, cytokines, risk genes, histology and serology metrics are associated with particular CTAPs. CTAPs are dynamic and can predict treatment response, highlighting the clinical utility of classifying rheumatoid arthritis synovial phenotypes. This comprehensive atlas and molecular, tissue-based stratification of rheumatoid arthritis synovial tissue reveal new insights into rheumatoid arthritis pathology and heterogeneity that could inform novel targeted treatments.

Immunopathogenesis of Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is the most common inflammatory arthropathy. The majority of evidence, derived from genetics, tissue analyses, models, and clinical studies, points to an immune-mediated etiology associated with stromal tissue dysregulation that together propogate chronic inflammation and articular destruction. A pre-RA phase lasting months to years may be characterized by the presence of circulating autoantibodies, increasing concentration and range of inflammatory cytokines and chemokines, and altered metabolism. Clinical disease onset comprises synovitis and systemic comorbidities affecting the vasculature, metabolism, and bone. Targeted immune therapeutics and aggressive treatment strategies have substantially improved clinical outcomes and informed pathogenetic understanding, but no cure as yet exists. Herein we review recent data that support intriguing models of disease pathogenesis. They allude to the possibility of restoration of immunologic homeostasis and thus a state of tolerance associated with drug-free remission. This target represents a bold vision for the future of RA therapeutics.

IgG Epitopes Processed and Presented by IgG+ B Cells Induce Suppression by Human Thymic-Derived Regulatory T Cells.

We described a human regulatory T cell (Treg) population activated by IgG+ B cells presenting peptides of the heavy C region (Fc) via processing of the surface IgG underlying a model for B cell-Treg cooperation in the human immune regulation. Functionally, Treg inhibited the polarization of naive T cells toward a proinflammatory phenotype in both a cognate and a noncognate fashion. Their fine specificities were similar in healthy donors and patients with rheumatoid arthritis, a systemic autoimmune disease. Four immunodominant Fc peptides bound multiple HLA class II alleles and were recognized by most subjects in the two cohorts. The presentation of Fc peptides that stimulate Treg through the processing of IgG by dendritic cells (DC) occurred in myeloid DC classical DC 1 and classical DC 2. Different routes of Ag processing of the IgG impacted Treg expansion in rheumatoid arthritis patients.

Persistent Joint Pain Following Arthropod Virus Infections.

PURPOSE OF REVIEW: Persistent joint pain is a common manifestation of arthropod-borne viral infections and can cause long-term disability. We review the epidemiology, pathophysiology, diagnosis, and management of arthritogenic alphavirus infection. RECENT FINDINGS: The global re-emergence of alphaviral outbreaks has led to an increase in virus-induced arthralgia and arthritis. Alphaviruses, including Chikungunya, O'nyong'nyong, Sindbis, Barmah Forest, Ross River, and Mayaro viruses, are associated with acute and/or chronic rheumatic symptoms. Identification of Mxra8 as a viral entry receptor in the alphaviral replication pathway creates opportunities for treatment and prevention. Recent evidence suggesting virus does not persist in synovial fluid during chronic chikungunya infection indicates that immunomodulators may be given safely. The etiology of persistent joint pain after alphavirus infection is still poorly understood. New diagnostic tools along and evidence-based treatment could significantly improve morbidity and long-term disability.

PTPN14 phosphatase and YAP promote TGFß signalling in rheumatoid synoviocytes.

OBJECTIVE: We aimed to understand the role of the tyrosine phosphatase PTPN14-which in cancer cells modulates the Hippo pathway by retaining YAP in the cytosol-in fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA). METHODS: Gene/protein expression levels were measured by quantitative PCR and/or Western blotting. Gene knockdown in RA FLS was achieved using antisense oligonucleotides. The interaction between PTPN14 and YAP was assessed by immunoprecipitation. The cellular localisation of YAP and SMAD3 was examined via immunofluorescence. SMAD reporter studies were carried out in HEK293T cells. The RA FLS/cartilage coimplantation and passive K/BxN models were used to examine the role of YAP in arthritis. RESULTS: RA FLS displayed overexpression of PTPN14 when compared with FLS from patients with osteoarthritis (OA). PTPN14 knockdown in RA FLS impaired TGFß-dependent expression of MMP13 and potentiation of TNF signalling. In RA FLS, PTPN14 formed a complex with YAP. Expression of PTPN14 or nuclear YAP-but not of a non-YAP-interacting PTPN14 mutant-enhanced SMAD reporter activity. YAP promoted TGFß-dependent SMAD3 nuclear localisation in RA FLS. Differences in epigenetic marks within Hippo pathway genes, including YAP, were found between RA FLS and OA FLS. Inhibition of YAP reduced RA FLS pathogenic behaviour and ameliorated arthritis severity. CONCLUSION: In RA FLS, PTPN14 and YAP promote nuclear localisation of SMAD3. YAP enhances a range of RA FLS pathogenic behaviours which, together with epigenetic evidence, points to the Hippo pathway as an important regulator of RA FLS behaviour.

Toreforant, an orally active histamine H4-receptor antagonist, in patients with active rheumatoid arthritis despite methotrexate: mechanism of action results from a phase 2, multicenter, randomized, double-blind, placebo-controlled synovial biopsy study.

OBJECTIVE/DESIGN: In a double-blind, placebo-controlled, multiple-dose study, we assessed the molecular mechanism of action of the selective histamine-4-receptor antagonist toreforant. PATIENTS/TREATMENT: Patients with active rheumatoid arthritis (RA) despite methotrexate were randomized (3:1) to toreforant 30 mg/day (weeks 0-52) or placebo (weeks 0-12) followed by toreforant 30 mg/day (weeks 12-52). METHODS: Primary biomarker analyses comprised 39 different proteins/mRNA transcripts measured in synovial biopsy (n = 39) and/or time-matched serum (n = 15) samples collected at baseline and week 6. Clinical response was assessed using C-reactive protein-based 28-joint disease activity scores. Data were summarized using descriptive statistics. RESULTS: Among 21 randomized, treated patients (toreforant-16, placebo-5), 18 (toreforant-13, placebo-5) completed the 12-week double-blind period (none completed open-label treatment) prior to the early study termination. Biomarker profiling indicated potential modest effects of toreforant on gene expression of histamine-1-receptor, tumor necrosis factor-alpha, and interleukin-8 in synovium. Potential trends between biomarkers and clinical response were observed with synovial monocyte chemoattractant protein-4 and phosphorylated extracellular-signal-regulated kinases and serum matrix metalloproteinase-3. Minimal synovial gene expression of interleukins-17A and 17F was detected. CONCLUSIONS: While clear biomarker signals associated with toreforant pharmacology in RA patients were not identified, modest associations between biomarkers and clinical response were noted. Synovial expression of interleukins-17A/17F was minimal. Limited sample size warrants cautious interpretation.

Regulation of the Cell Cycle and Inflammatory Arthritis by the Transcription Cofactor LBH Gene.

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-1ß 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.

Epigenetics of inflammatory arthritis.

PURPOSE OF REVIEW: Aberrant epigenetic changes in DNA methylation, histone marks, and noncoding RNA expression regulate the pathogenesis of many rheumatic diseases. The present article will review the recent advances in the epigenetic profile of inflammatory arthritis and discuss diagnostic biomarkers and potential therapeutic targets. RECENT FINDINGS: Methylation signatures of fibroblast-like synoviocytes not only distinguish rheumatoid arthritis (RA) and osteoarthritis (OA), but also early RA from late RA or juvenile idiopathic arthritis. Methylation patterns are also specific to individual joint locations, which might explain the distribution of joint involvement in some rheumatic diseases. Hypomethylation in systemic lupus erythematosus (SLE) T cells is, in part, because of active demethylation and 5-hydroxymethylation. The methylation status of some genes in SLE is associated with disease severity and has potential as a diagnostic marker. An integrative analysis of OA methylome, transcriptome, and proteome in chondrocytes has identified multiple-evidence genes that might be evaluated for therapeutic potential. Class-specific histone deacetylase inhibitors are being evaluated for therapy in inflammatory arthritis. SUMMARY: Disease pathogenesis is regulated by the interplay of genetics, environment, and epigenetics. Understanding how these mechanisms regulate cell function in health and disease has implications for individualized therapy.

Hexokinase 2 as a novel selective metabolic target for rheumatoid arthritis.

OBJECTIVES: Recent studies indicate that glucose metabolism is altered in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS). Hexokinases (HKs) catalyse the first step in glucose metabolism, and HK2 constitutes the principal HK inducible isoform. We hypothesise that HK2 contributes to the synovial lining hypertrophy and plays a critical role in bone and cartilage damage. METHODS: HK1 and HK2 expression were determined in RA and osteoarthritis (OA) synovial tissue by immunohistochemistry. RA FLS were transfected with either HK1 or HK2 siRNA, or infected with either adenovirus (ad)-GFP, ad-HK1 or ad-HK2. FLS migration and invasion were assessed. To study the role of HK2 in vivo, 108 particles of ad-HK2 or ad-GFP were injected into the knee of wild-type mice. K/BxN serum transfer arthritis was induced in HK2F/F mice harbouring Col1a1-Cre (HK2Col1), to delete HK2 in non-haematopoietic cells. RESULTS: HK2 is particular of RA histopathology (9/9 RA; 1/8 OA) and colocalises with FLS markers. Silencing HK2 in RA FLS resulted in a less invasive and migratory phenotype. Consistently, overexpression of HK2 resulted in an increased ability to migrate and invade. It also increased extracellular lactate production. Intra-articular injection of ad-HK2 in normal knees dramatically increased synovial lining thickness, FLS activation and proliferation. HK2 was highly expressed in the synovial lining after K/BxN serum transfer arthritis. HK2Col1 mice significantly showed decreased arthritis severity, bone and cartilage damage. CONCLUSION: HK2 is specifically expressed in RA synovial lining and regulates FLS aggressive functions. HK2 might be an attractive selective metabolic target safer than global glycolysis for RA treatment.

Inflammasome-mediated disease animal models reveal roles for innate but not adaptive immunity.

NLRP3 nucleates the inflammasome, a protein complex responsible for cleavage of prointerleukin-1beta (IL-1beta) to its active form. Mutations in the NLRP3 gene cause the autoinflammatory disease spectrum cryopyrin-associated periodic syndromes (CAPS). The central role of IL-1beta in CAPS is supported by the response to IL-1-targeted therapy. We developed two Nlrp3 mutant knockin mouse strains to model CAPS to examine the role of other inflammatory mediators and adaptive immune responses in an innate immune-driven disease. These mice had systemic inflammation and poor growth, similar to some human CAPS patients, and demonstrated early mortality, primarily mediated by myeloid cells. Mating these mutant mice to various gene mutant backgrounds showed that the mouse disease phenotype required an intact inflammasome, was only partially dependent on IL-1beta, and was independent of T cells. These data suggest that CAPS are true inflammasome-mediated diseases and provide insight for more common inflammatory disorders.

PATHOGENESIS OF RHEUMATOID ARTHRITIS: THE INTERSECTION OF GENETICS AND EPIGENETICS.

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.

Computationally expanding infinium HumanMethylation450 BeadChip array data to reveal distinct DNA methylation patterns of rheumatoid arthritis.

MOTIVATION: DNA methylation signatures in rheumatoid arthritis (RA) have been identified in fibroblast-like synoviocytes (FLS) with Illumina HumanMethylation450 array. Since <2% of CpG sites are covered by the Illumina 450K array and whole genome bisulfite sequencing is still too expensive for many samples, computationally predicting DNA methylation levels based on 450K data would be valuable to discover more RA-related genes. RESULTS: We developed a computational model that is trained on 14 tissues with both whole genome bisulfite sequencing and 450K array data. This model integrates information derived from the similarity of local methylation pattern between tissues, the methylation information of flanking CpG sites and the methylation tendency of flanking DNA sequences. The predicted and measured methylation values were highly correlated with a Pearson correlation coefficient of 0.9 in leave-one-tissue-out cross-validations. Importantly, the majority (76%) of the top 10% differentially methylated loci among the 14 tissues was correctly detected using the predicted methylation values. Applying this model to 450K data of RA, osteoarthritis and normal FLS, we successfully expanded the coverage of CpG sites 18.5-fold and accounts for about 30% of all the CpGs in the human genome. By integrative omics study, we identified genes and pathways tightly related to RA pathogenesis, among which 12 genes were supported by triple evidences, including 6 genes already known to perform specific roles in RA and 6 genes as new potential therapeutic targets. AVAILABILITY AND IMPLEMENTATION: The source code, required data for prediction, and demo data for test are freely available at: http://wanglab.ucsd.edu/star/LR450K/ CONTACT: wei-wang@ucsd.edu or gfirestein@ucsd.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

TGFß responsive tyrosine phosphatase promotes rheumatoid synovial fibroblast invasiveness.

OBJECTIVE: In rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) that line joint synovial membranes aggressively invade the extracellular matrix, destroying cartilage and bone. As signal transduction in FLS is mediated through multiple pathways involving protein tyrosine phosphorylation, we sought to identify protein tyrosine phosphatases (PTPs) regulating the invasiveness of RA FLS. We describe that the transmembrane receptor PTPκ (RPTPκ), encoded by the transforming growth factor (TGF) ß-target gene, PTPRK, promotes RA FLS invasiveness. METHODS: Gene expression was quantified by quantitative PCR. PTP knockdown was achieved using antisense oligonucleotides. FLS invasion and migration were assessed in transwell or spot assays. FLS spreading was assessed by immunofluorescence microscopy. Activation of signalling pathways was analysed by Western blotting of FLS lysates using phosphospecific antibodies. In vivo FLS invasiveness was assessed by intradermal implantation of FLS into nude mice. The RPTPκ substrate was identified by pull-down assays. RESULTS: PTPRK expression was higher in FLS from patients with RA versus patients with osteoarthritis, resulting from increased TGFB1 expression in RA FLS. RPTPκ knockdown impaired RA FLS spreading, migration, invasiveness and responsiveness to platelet-derived growth factor, tumour necrosis factor and interleukin 1 stimulation. Furthermore, RPTPκ deficiency impaired the in vivo invasiveness of RA FLS. Molecular analysis revealed that RPTPκ promoted RA FLS migration by dephosphorylation of the inhibitory residue Y527 of SRC. CONCLUSIONS: By regulating phosphorylation of SRC, RPTPκ promotes the pathogenic action of RA FLS, mediating cross-activation of growth factor and inflammatory cytokine signalling by TGFß in RA FLS.

Phosphoinositide 3-kinase δ regulates migration and invasion of synoviocytes in rheumatoid arthritis.

Cartilage destruction mediated by invasive fibroblast-like synoviocytes (FLS) plays a central role in pathogenesis of rheumatoid arthritis (RA). Increased cell migration and degradation of extracellular matrix are fundamental to these processes. The class I PI3Ks control cell survival, proliferation, and migration, which might be involved in cartilage damage in RA. PI3Kδ isoform was recently identified as a key regulator of FLS growth and survival, suggesting that it could contribute to synoviocyte aggressive behavior. Therefore, we assessed the role of PI3Kδ in RA synoviocyte migration and invasion. We observed that PI3Kδ inhibition or small interfering RNA knockdown decreased platelet-derived growth factor (PDGF)-mediated migration and invasion of FLS. We then showed that PI3Kδ regulates the organization of actin cytoskeleton and lamellipodium formation during PDGF stimulation. To gain insight into molecular mechanisms, we examined the effect of PI3Kδ inhibition on Rac1/PAK, FAK, and JNK activation. Our studies suggest that Rac1/PAK is key target of PDGF-mediated PI3Kδ signaling, whereas FAK and JNK are not involved. Thus, PI3Kδ contributes to multiple aspects of the pathogenic FLS behavior in RA. These observations, together with previous findings that PI3Kδ regulates FLS growth and survival, suggest that PI3Kδ inhibition could be chondroprotective in RA by modulating synoviocyte growth, migration, and invasion.

Anti-Inflammatory Effects and Joint Protection in Collagen-Induced Arthritis after Treatment with IQ-1S, a Selective c-Jun N-Terminal Kinase Inhibitor.

c-Jun N-terminal kinases (JNKs) participate in many physiologic and pathologic processes, including inflammatory diseases. We recently synthesized the sodium salt of IQ-1S (11H-indeno[1,2-b]quinoxalin-11-one oxime) and demonstrated that it is a high-affinity JNK inhibitor and inhibits murine delayed-type hypersensitivity. Here we show that IQ-1S is highly specific for JNK and that its neutral form is the most abundant species at physiologic pH. Molecular docking of the IQ-1S syn isomer into the JNK1 binding site gave the best pose, which corresponded to the position of cocrystallized JNK inhibitor SP600125 (1,9-pyrazoloanthrone). Evaluation of the therapeutic potential of IQ-1S showed that it inhibited matrix metalloproteinase 1 and 3 gene expression induced by interleukin-1ß in human fibroblast-like synoviocytes and significantly attenuated development of murine collagen-induced arthritis (CIA). Treatment with IQ-1S either before or after induction of CIA resulted in decreased clinical scores, and joint sections from IQ-1S-treated CIA mice exhibited only mild signs of inflammation and minimal cartilage loss compared with those from control mice. Collagen II-specific antibody responses were also reduced by IQ-1S treatment. By contrast, the inactive ketone derivative 11H-indeno[1,2-b]quinoxalin-11-one had no effect on CIA clinical scores or collagen II-specific antibody titers. IQ-1S treatment also suppressed proinflammatory cytokine and chemokine levels in joints and lymph node cells. Finally, treatment with IQ-1S increased the number of Foxp3(+)CD4(+)CD25(+) regulatory T cells in lymph nodes. Thus, IQ-1S can reduce inflammation and cartilage loss associated with CIA and can serve as a small-molecule modulator for mechanistic studies of JNK function in rheumatoid arthritis.