Single-cell analysis reveals key differences between early-stage and late-stage systemic sclerosis skin across autoantibody subgroups.

OBJECTIVES: The severity of skin involvement in diffuse cutaneous systemic sclerosis (dcSSc) depends on stage of disease and differs between anti-RNA-polymerase III (ARA) and anti-topoisomerase antibody (ATA) subsets. We have investigated cellular differences in well-characterised dcSSc patients compared with healthy controls (HCs). METHODS: We performed single-cell RNA sequencing on 4 mm skin biopsy samples from 12 patients with dcSSc and HCs (n=3) using droplet-based sequencing (10× genomics). Patients were well characterised by stage (>5 or <5 years disease duration) and autoantibody (ATA+ or ARA+). Analysis of whole skin cell subsets and fibroblast subpopulations across stage and ANA subgroup were used to interpret potential cellular differences anchored by these subgroups. RESULTS: Fifteen forearm skin biopsies were analysed. There was a clear separation of SSc samples, by disease, stage and antibody, for all cells and fibroblast subclusters. Further analysis revealed differing cell cluster gene expression profiles between ATA+ and ARA+ patients. Cell-to-cell interaction suggest differing interactions between early and late stages of disease and autoantibody. TGFß response was mainly seen in fibroblasts and smooth muscle cells in early ATA+dcSSc skin samples, whereas in early ARA+dcSSc patient skin samples, the responding cells were endothelial, reflect broader differences between clinical phenotypes and distinct skin score trajectories across autoantibody subgroups of dcSSc. CONCLUSIONS: We have identified cellular differences between the two main autoantibody subsets in dcSSc (ARA+ and ATA+). These differences reinforce the importance of considering autoantibody and stage of disease in management and trial design in SSc.

Phosphatidylinositol 3-kinase delta pathway: a novel therapeutic target for Sjögren's syndrome.

BACKGROUND: The phosphatidylinositol 3-kinase delta isoform (PI3Kδ) belongs to an intracellular lipid kinase family that regulate lymphocyte metabolism, survival, proliferation, apoptosis and migration and has been successfully targeted in B-cell malignancies. Primary Sjögren's syndrome (pSS) is a chronic immune-mediated inflammatory disease characterised by exocrine gland lymphocytic infiltration and B-cell hyperactivation which results in systemic manifestations, autoantibody production and loss of glandular function. Given the central role of B cells in pSS pathogenesis, we investigated PI3Kδ pathway activation in pSS and the functional consequences of blocking PI3Kδ in a murine model of focal sialoadenitis that mimics some features of pSS. METHODS AND RESULTS: Target validation assays showed significant expression of phosphorylated ribosomal protein S6 (pS6), a downstream mediator of the phosphatidylinositol 3-kinase delta (PI3Kδ) pathway, within pSS salivary glands. pS6 distribution was found to co-localise with T/B cell markers within pSS aggregates and the CD138+ plasma cells infiltrating the glands. In vivo blockade of PI3Kδ activity with seletalisib, a PI3Kδ-selective inhibitor, in a murine model of focal sialoadenitis decreased accumulation of lymphocytes and plasma cells within the glands of treated mice in the prophylactic and therapeutic regimes. Additionally, production of lymphoid chemokines and cytokines associated with ectopic lymphoneogenesis and, remarkably, saliva flow and autoantibody production, were significantly affected by treatment with seletalisib. CONCLUSION: These data demonstrate activation of PI3Kδ pathway within the glands of patients with pSS and its contribution to disease pathogenesis in a model of disease, supporting the exploration of the therapeutic potential of PI3Kδ pathway inhibition in this condition.

Targeting early changes in the synovial microenvironment: a new class of immunomodulatory therapy?

OBJECTIVES: Controlled immune responses rely on integrated crosstalk between cells and their microenvironment. We investigated whether targeting proinflammatory signals from the extracellular matrix that persist during pathological inflammation provides a viable strategy to treat rheumatoid arthritis (RA). METHODS: Monoclonal antibodies recognising the fibrinogen-like globe (FBG) of tenascin-C were generated by phage display. Clones that neutralised FBG activation of toll-like receptor 4 (TLR4), without impacting pathogenic TLR4 activation, were epitope mapped by crystallography. Antibodies stained synovial biopsies of patients at different stages of RA development. Antibody efficacy in preventing RA synovial cell cytokine release, and in modulating collagen-induced arthritis in rats, was assessed. RESULTS: Tenascin-C is expressed early in the development of RA, even before disease diagnosis, with higher levels in the joints of people with synovitis who eventually developed RA than in people whose synovitis spontaneously resolved. Anti-FBG antibodies inhibited cytokine release by RA synovial cells and prevented disease progression and tissue destruction during collagen-induced arthritis. CONCLUSIONS: Early changes in the synovial microenvironment contribute to RA progression; blocking proinflammatory signals from the matrix can ameliorate experimental arthritis. These data highlight a new drug class that could offer early, disease-specific immune modulation in RA, without engendering global immune suppression.

Synovial cellular and molecular signatures stratify clinical response to csDMARD therapy and predict radiographic progression in early rheumatoid arthritis patients.

OBJECTIVES: To unravel the hierarchy of cellular/molecular pathways in the disease tissue of early, treatment-naïve rheumatoid arthritis (RA) patients and determine their relationship with clinical phenotypes and treatment response/outcomes longitudinally. METHODS: 144 consecutive treatment-naïve early RA patients (<12 months symptoms duration) underwent ultrasound-guided synovial biopsy before and 6 months after disease-modifying antirheumatic drug (DMARD) initiation. Synovial biopsies were analysed for cellular (immunohistology) and molecular (NanoString) characteristics and results compared with clinical and imaging outcomes. Differential gene expression analysis and logistic regression were applied to define variables correlating with treatment response and predicting radiographic progression. RESULTS: Cellular and molecular analyses of synovial tissue demonstrated for the first time in early RA the presence of three pathology groups: (1) lympho-myeloid dominated by the presence of B cells in addition to myeloid cells; (2) diffuse-myeloid with myeloid lineage predominance but poor in B cells nd (3) pauci-immune characterised by scanty immune cells and prevalent stromal cells. Longitudinal correlation of molecular signatures demonstrated that elevation of myeloid- and lymphoid-associated gene expression strongly correlated with disease activity, acute phase reactants and DMARD response at 6 months. Furthermore, elevation of synovial lymphoid-associated genes correlated with autoantibody positivity and elevation of osteoclast-targeting genes predicting radiographic joint damage progression at 12 months. Patients with predominant pauci-immune pathology showed less severe disease activity and radiographic progression. CONCLUSIONS: We demonstrate at disease presentation, prior to pathology modulation by therapy, the presence of specific cellular/molecular synovial signatures that delineate disease severity/progression and therapeutic response and may pave the way to more precise definition of RA taxonomy, therapeutic targeting and improved outcomes.

Identification of a transitional fibroblast function in very early rheumatoid arthritis.

OBJECTIVES: Synovial fibroblasts actively regulate the inflammatory infiltrate by communicating with neighbouring endothelial cells (EC). Surprisingly, little is known about how the development of rheumatoid arthritis (RA) alters these immunomodulatory properties. We examined the effects of phase of RA and disease outcome (resolving vs persistence) on fibroblast crosstalk with EC and regulation of lymphocyte recruitment. METHODS: Fibroblasts were isolated from patients without synovitis, with resolving arthritis, very early RA (VeRA; symptom ≤12 weeks) and established RA undergoing joint replacement (JRep) surgery. Endothelial-fibroblast cocultures were formed on opposite sides of porous filters. Lymphocyte adhesion from flow, secretion of soluble mediators and interleukin 6 (IL-6) signalling were assessed. RESULTS: Fibroblasts from non-inflamed and resolving arthritis were immunosuppressive, inhibiting lymphocyte recruitment to cytokine-treated endothelium. This effect was lost very early in the development of RA, such that fibroblasts no longer suppressed recruitment. Changes in IL-6 and transforming growth factor beta 1 (TGF-ß1) signalling appeared critical for the loss of the immunosuppressive phenotype. In the absence of exogenous cytokines, JRep, but not VeRA, fibroblasts activated endothelium to support lymphocyte. CONCLUSIONS: In RA, fibroblasts undergo two distinct changes in function: first a loss of immunosuppressive responses early in disease development, followed by the later acquisition of a stimulatory phenotype. Fibroblasts exhibit a transitional functional phenotype during the first 3 months of symptoms that contributes to the accumulation of persistent infiltrates. Finally, the role of IL-6 and TGF-ß1 changes from immunosuppressive in resolving arthritis to stimulatory very early in the development of RA. Early interventions targeting 'pathogenic' fibroblasts may be required in order to restore protective regulatory processes.

Inflammation across tissues: can shared cell biology help design smarter trials?

Immune-mediated inflammatory diseases (IMIDs) are responsible for substantial global disease burden and associated health-care costs. Traditional models of research and service delivery silo their management within organ-based medical disciplines. Very often patients with disease in one organ have comorbid involvement in another, suggesting shared pathogenic pathways. Moreover, different IMIDs are often treated with the same drugs (including glucocorticoids, immunoregulators and biologics). Unlocking the cellular basis of these diseases remains a major challenge, leading us to ask why, if these diseases have so much in common, they are not investigated in a common manner. A tissue-based, cellular understanding of inflammation might pave the way for cross-disease, cross-discipline basket trials (testing one drug across two or more diseases) to reduce the risk of failure of early-phase drug development in IMIDs. This new approach will enable rapid assessment of the efficacy of new therapeutic agents in cross-disease translational research in humans.

Metabolic Checkpoints in Rheumatoid Arthritis.

Several studies have highlighted the interplay between metabolism, immunity and inflammation. Both tissue resident and infiltrating immune cells play a major role in the inflammatory process of rheumatoid arthritis (RA) via the production of cytokines, adipo-cytokines and metabolic intermediates. These functions are metabolically demanding and require the most efficient use of bioenergetic pathways. The synovial membrane is the primary site of inflammation in RA and exhibits distinctive histological patterns characterized by different metabolism, prognosis and response to treatment. In the RA synovium, the high energy demand by stromal and infiltrating immune cells, causes the accumulation of metabolites, and adipo-cytokines, which carry out signaling functions, as well as activating transcription factors which act as metabolic sensors. These events drive immune and joint-resident cells to acquire pro-inflammatory effector functions which in turn perpetuate chronic inflammation. Whether metabolic changes are a consequence of the disease or one of the causes of RA pathogenesis is still under investigation. This review covers our current knowledge of cell metabolism in RA. Understanding the intricate interactions between metabolic pathways and the inflammatory and immune responses will provide more awareness of the mechanisms underlying RA pathogenesis and will identify novel therapeutic options to treat this disease.

Genetic Deletion of the Stromal Cell Marker CD248 (Endosialin) Protects against the Development of Renal Fibrosis.

BACKGROUND: Tissue fibrosis and microvascular rarefaction are hallmarks of progressive renal disease. CD248 is a transmembrane glycoprotein expressed by key effector cells within the stroma of fibrotic kidneys including pericytes, myofibroblasts and stromal fibroblasts. In human disease, increased expression of CD248 by stromal cells predicts progression to end-stage renal failure. We therefore, hypothesized that the genetic deletion of the CD248 gene would protect against fibrosis following kidney injury. METHODS: Using the unilateral ureteral obstruction (UUO) model of renal fibrosis, we investigated the effect of genetic deletion of CD248 on post obstructive kidney fibrosis. RESULTS: CD248 null mice were protected from fibrosis and microvascular rarefaction following UUO. Although the precise mechanism is not known, this may to be due to a stabilizing effect of pericytes with less migration and differentiation of pericytes toward a myofibroblast phenotype in CD248-/- mice. CD248-/- fibroblasts also proliferated less and deposited less collagen in vitro. CONCLUSION: These studies suggest that CD248 stromal cells have a pathogenic role in renal fibrosis and that targeting CD248 is effective at inhibiting both microvascular rarefaction and renal fibrosis through modulation of pericyte and stromal cell function.