Computational approaches in rheumatic diseases - Deciphering complex spatio-temporal cell interactions.

Inflammatory arthritis, including rheumatoid (RA), and psoriatic (PsA) arthritis, are clinically and immunologically heterogeneous diseases with no identified cure. Chronic inflammation of the synovial tissue ushers loss of function of the joint that severely impacts the patient's quality of life, eventually leading to disability and life-threatening comorbidities. The pathogenesis of synovial inflammation is the consequence of compounded immune and stromal cell interactions influenced by genetic and environmental factors. Deciphering the complexity of the synovial cellular landscape has accelerated primarily due to the utilisation of bulk and single cell RNA sequencing. Particularly the capacity to generate cell-cell interaction networks could reveal evidence of previously unappreciated processes leading to disease. However, there is currently a lack of universal nomenclature as a result of varied experimental and technological approaches that discombobulates the study of synovial inflammation. While spatial transcriptomic analysis that combines anatomical information with transcriptomic data of synovial tissue biopsies promises to provide more insights into disease pathogenesis, in vitro functional assays with single-cell resolution will be required to validate current bioinformatic applications. In order to provide a comprehensive approach and translate experimental data to clinical practice, a combination of clinical and molecular data with machine learning has the potential to enhance patient stratification and identify individuals at risk of arthritis that would benefit from early therapeutic intervention. This review aims to provide a comprehensive understanding of the effect of computational approaches in deciphering synovial inflammation pathogenesis and discuss the impact that further experimental and novel computational tools may have on therapeutic target identification and drug development.

Distinct stromal and immune cell interactions shape the pathogenesis of rheumatoid and psoriatic arthritis.

OBJECTIVES: Immune and stromal cell communication is central in the pathogenesis of rheumatoid arthritis (RA) and psoriatic arthritis (PsA), however, the nature of these interactions in the synovial pathology of the two pathotypes can differ. Identifying immune-stromal cell crosstalk at the site of inflammation in RA and PsA is challenging. This study creates the first global transcriptomic analysis of the RA and PsA inflamed joint and investigates immune-stromal cell interactions in the pathogenesis of synovial inflammation. METHODS: Single cell transcriptomic profiling of 178 000 synovial tissue cells from five patients with PsA and four patients with RA, importantly, without prior sorting of immune and stromal cells. This approach enabled the transcriptomic analysis of the intact synovial tissue and identification of immune and stromal cell interactions. State of the art data integration and annotation techniques identified and characterised 18 stromal and 14 immune cell clusters. RESULTS: Global transcriptomic analysis of synovial cell subsets identifies actively proliferating synovial T cells and indicates that due to differential λ and κ immunoglobulin light chain usage, synovial plasma cells are potentially not derived from the local memory B cell pool. Importantly, we report distinct fibroblast and endothelial cell transcriptomes indicating abundant subpopulations in RA and PsA characterised by differential transcription factor usage. Using receptor-ligand interactions and downstream target characterisation, we identify RA-specific synovial T cell-derived transforming growth factor (TGF)-ß and macrophage interleukin (IL)-1ß synergy in driving the transcriptional profile of FAPα+THY1+ invasive synovial fibroblasts, expanded in RA compared with PsA. In vitro characterisation of patient with RA synovial fibroblasts showed metabolic switch to glycolysis, increased adhesion intercellular adhesion molecules 1 expression and IL-6 secretion in response to combined TGF-ß and IL-1ß treatment. Disrupting specific immune and stromal cell interactions offers novel opportunities for targeted therapeutic intervention in RA and PsA.

First use of tofacitinib to treat an immune checkpoint inhibitor-induced arthritis.

Immune checkpoint inhibitors have revolutionised cancer treatment; however, immune-related adverse events do occur, with up to 7% developing inflammatory arthritis. Common rheumatoid arthritis therapies such as methotrexate, prednisolone and biologics have been used to treat this arthritis in small, uncontrolled case series with varying success. In this case of personalised medicine, we report the first use of tofacitinib, a small molecular inhibitor of the Janus kinase-signal transducer and activator of transcription pathway, to treat checkpoint inhibitor-related inflammatory arthritis. This resulted in a rapid clinical response and complete, sustained remission of the arthritis with associated marked reduction in synovial molecular and cellular immune response.

The PD-1:PD-L1 axis in Inflammatory Arthritis.

The activation of antigen specific T cells during an immune response is a tightly regulated process at the level of both costimulatory and coinhibitory receptors. One such coinhibitory receptor or checkpoint inhibitor which has received much attention in the field of oncology is the programmed cell death protein 1 (PD-1). Blockade of PD-1 or its ligand PD-L1 has proven successful in the treatment of a wide variety of cancers, therefore highlighting an important role for this pathway in anti-tumour immune responses. However, a caveat of PD-1 therapy and boosting anti-tumour immune responses is the development of self-reactive T cells which can lead to the induction of various autoimmune or inflammatory diseases, referred to as immune- related adverse events (irAEs). The emergence of rheumatological irAEs such as Inflammatory Arthritis (IA) in recent years has highlighted the importance of PD-1 in maintaining self-tolerance. Furthermore, the emergence of rheumatology related irAEs raises an important question as to how defects in this pathway can contribute to spontaneous rheumatological disease. In this review, we describe the biological distribution, function and regulation of the PD-1 pathway, its potential role in IA and irAE related IA.

CD209/CD14+ Dendritic Cells Characterization in Rheumatoid and Psoriatic Arthritis Patients: Activation, Synovial Infiltration, and Therapeutic Targeting.

Dendritic cells (DC) have a key role in the initiation and progression of inflammatory arthritis (IA). In this study, we identified a DC population that derive from monocytes, characterized as CD209/CD14+ DC, expressing classical DC markers (HLADR, CD11c) and the Mo-DC marker (CD209), while also retaining the monocytic marker CD14. This CD209/CD14+ DC population is present in the circulation of Healthy Control (HC), with increased frequency in Rheumatoid Arthritis (RA) and Psoriatic arthritic (PsA) patients. We demonstrate, for the first time, that circulatory IA CD209/CD14+ DC express more cytokines (IL1ß/IL6/IL12/TNFα) and display a unique chemokine receptor expression and co-expression profiles compared to HC. We demonstrated that CD209/CD14+ DC are enriched in the inflamed joint where they display a unique inflammatory and maturation phenotype, with increased CD40 and CD80 and co-expression of specific chemokine receptors, displaying unique patterns between PsA and RA. We developed a new protocol of magnetic isolation and expansion for CD209+ DC from blood and identified transcriptional differences involved in endocytosis/antigen presentation between RA and PsA CD209+ DC. In addition, we observed that culture of healthy CD209+ DC with IA synovial fluid (SF), but not Osteoarthritis (OA) SF, was sufficient to induce the development of CD209/CD14+ DC, leading to a poly-mature DC phenotype. In addition, differential effects were observed in terms of chemokine receptor and chemokine expression, with healthy CD209+ DC displaying increased expression/co-expression of CCR6, CCR7, CXCR3, CXCR4 and CXCR5 when cultured with RA SF, while an increase in the chemokines CCR3, CXCL10 and CXCL11 was observed when cultured with PsA SF. This effect may be mediated in part by the observed differential increase in chemokines expressed in RA vs PsA SF. Finally, we observed that the JAK/STAT pathway, but not the NF-κB pathway (driven by TNFα), regulated CD209/CD14+ DC function in terms of activation, inflammatory state, and migratory capacity. In conclusion, we identified a novel CD209/CD14+ DC population, which is active in the circulation of RA and PsA, an effect potentiated once they enter the joint. Furthermore, we demonstrated that JAK/STAT inhibition can be used as a therapeutic strategy to decrease the inflammatory state of the pathogenic CD209/CD14+ DC.

Monocyte-Derived Dendritic Cell Differentiation in Inflammatory Arthritis Is Regulated by the JAK/STAT Axis via NADPH Oxidase Regulation.

Monocyte-derived Dendritic cells (Mo-DC) are a distinct DC subset, involved in inflammation and infection, they originate from monocytes upon stimulation in the circulation and their activation and function may vary in autoimmune diseases. In this study we investigate the differences in Mo-DC differentiation and function in patients with Rheumatoid (RA) compared to Psoriatic arthritis (PsA). A significant increase in the Mo-DC differentiation marker CD209, paralleled by a corresponding decrease in the monocytic marker CD14, was demonstrated in RA compared to PsA, as early as 1 day post Mo-DC differentiation. RA monocytes ex-vivo were phenotypically different to PsA, displaying a more mature phenotype associated with altered cellular-morphology, early dendrite formation, and a significant increase in the CD40 marker. In addition, SPICE algorithm flow cytometric analysis showed distinct differences in chemokine receptors distribution in HC compared to PsA and RA CD14+ cells in the blood, with increased expression of the chemokine receptors CCR7 and CXCR4 observed in PsA and RA. In addition CD14+ cells at the site of inflammation showed a different chemokine receptor pattern between PsA and RA patients, with higher expression of CXCR3 and CXCR5 in RA when compared to PsA. The early priming observed in RA resulted in monocyte-endocytosis and antigen-uptake mechanisms to be impaired, effects that were not observed in PsA where phagocytosis capacity remained highly functional. Tofacitinib inhibited early Mo-DC differentiation, decreasing both CD209 and CD40 activation markers in RA. Inhibition of Mo-DC differentiation in response to Tofacitinib was mediated via an imbalance in the activation of NADPH-oxidases NOX5 and NOX2. This effect was reversed by NOX5 inhibition, but not NOX2, resulting in suppression of NOX5-dependent ROS production. In conclusion, RA monocytes are already primed ex vivo to become DC, evident by increased expression of activation markers, morphological appearance and impaired endocytosis capacity. Furthermore, we demonstrated for the first time that NOX5 mediates Mo-DC differentiation and function in response to Tofacitinib, which may alter DC functions.

Low Density Granulocytes in ANCA Vasculitis Are Heterogenous and Hypo-Responsive to Anti-Myeloperoxidase Antibodies.

Low Density Granulocytes (LDGs), which appear in the peripheral blood mononuclear cell layer of density-separated blood, are seen in cancer, sepsis, autoimmunity, and pregnancy. Their significance in ANCA vasculitis (AAV) is little understood. As these cells bear the autoantigens associated with this condition and have been found to undergo spontaneous NETosis in other diseases, we hypothesized that they were key drivers of vascular inflammation. We found that LDGs comprise a 3-fold higher fraction of total granulocytes in active vs. remission AAV and disease controls. They are heterogeneous, split between cells displaying mature (75%), and immature (25%) phenotypes. Surprisingly, LDGs (unlike normal density granulocytes) are hyporesponsive to anti-myeloperoxidase antibody stimulation, despite expressing myeloperoxidase on their surface. They are characterized by reduced CD16, CD88, and CD10 expression, higher LOX-1 expression and immature nuclear morphology. Reduced CD16 expression is like that observed in the LDG population in umbilical cord blood and in granulocytes of humanized mice treated with G-CSF. LDGs in AAV are thus a mixed population of mature and immature neutrophils. Their poor response to anti-MPO stimulation suggests that, rather than being a primary driver of AAV pathogenesis, LDGs display characteristics consistent with generic emergency granulopoiesis responders in the context of acute inflammation.

Spontaneous atopic dermatitis in mice with a defective skin barrier is independent of ILC2 and mediated by IL-1ß.

BACKGROUND: Atopic dermatitis (AD) is one of the most common skin diseases with a multifactorial etiology. Mutations leading to loss of skin barrier function are associated with the development of AD with group 2 innate lymphoid cells (ILC2) promoting acute skin inflammation. Filaggrin-mutant (Flgft/ft ) mice develop spontaneous skin inflammation accompanied by an increase in skin ILC2 numbers, IL-1ß production, and other cytokines recapitulating human AD. Here, we investigated the role of ILC2, effector cytokines, inflammasome activation, and mast cell function on the development of chronic AD-like inflammation in mice. METHODS: Mice with a frameshift mutation in the filaggrin gene develop spontaneous dermatitis. Flgft/ft mice were crossed to cell- or cytokine-deficient mouse strains, or bred under germ-free conditions. Skin inflammation was scored, and microbiome composition was analyzed. Skin protein expression was measured by multiplex immunoassay. Infiltrating cells were analyzed by flow cytometry. RESULTS: Wild-type and Flgft/ft mice significantly differ in their microbiome composition. Furthermore, mutant mice do not develop skin inflammation under germ-free conditions. ILC2 deficiency did not ameliorate chronic dermatitis in Flgft/ft mice, which was also independent of IL-4, IL-5, IL-9, IL-13, IL-17A, and IL-22. Inflammation was independent of NLRP3 inflammasome activation but required IL-1ß and IL-1R1-signaling. Mechanistically, IL-1ß promoted hyperactivation of IL-1R1-expressing mast cells. Treatment with anti-IL-1ß-antibody alleviated dermatitis exacerbation, while antibiotic intervention ameliorated dermatitis in neonatal mice but not in adults with established inflammation. CONCLUSIONS: In summary, we identified a critical role for the microbiome and IL-1ß mediating chronic inflammation in mice with an impaired skin barrier.

IL-17 Receptor A Maintains and Protects the Skin Barrier To Prevent Allergic Skin Inflammation.

Atopic dermatitis (AD) is a common inflammatory skin disease affecting up to 20% of children and 3% of adults worldwide and is associated with dysregulation of the skin barrier. Although type 2 responses are implicated in AD, emerging evidence indicates a potential role for the IL-17A signaling axis in AD pathogenesis. In this study we show that in the filaggrin mutant mouse model of spontaneous AD, IL-17RA deficiency (Il17ra-/- ) resulted in severe exacerbation of skin inflammation. Interestingly, Il17ra-/- mice without the filaggrin mutation also developed spontaneous progressive skin inflammation with eosinophilia, as well as increased levels of thymic stromal lymphopoietin (TSLP) and IL-5 in the skin. Il17ra-/- mice have a defective skin barrier with altered filaggrin expression. The barrier dysregulation and spontaneous skin inflammation in Il17ra-/- mice was dependent on TSLP, but not the other alarmins IL-25 and IL-33. The associated skin inflammation was mediated by IL-5-expressing pathogenic effector Th2 cells and was independent of TCRγδ T cells and IL-22. An absence of IL-17RA in nonhematopoietic cells, but not in the hematopoietic cells, was required for the development of spontaneous skin inflammation. Skin microbiome dysbiosis developed in the absence of IL-17RA, with antibiotic intervention resulting in significant amelioration of skin inflammation and reductions in skin-infiltrating pathogenic effector Th2 cells and TSLP. This study describes a previously unappreciated protective role for IL-17RA signaling in regulation of the skin barrier and maintenance of skin immune homeostasis.

PD-L1hi B cells are critical regulators of humoral immunity.

Specific B-cell subsets can regulate T-cell immune responses, and are termed regulatory B cells (Breg). The majority of Breg cells described in mouse and man have been identified by IL-10 production and are known to suppress allergy and autoimmunity. However, Breg cell mediated immune suppression, independent of IL-10, also occurs. Here we show that Breg cells play a critical role in regulating humoral immunity mediated by CD4(+)CXCR5(+)PD-1(+) follicular helper T cells, and can suppress inflammation in autoimmune disease through elevated expression of PD-L1. We have also identified that these B cells are resistant to αCD20 B-cell depletion. This work describes how Breg cells are critical in humoral homoeostasis and may have implications for the regulation of autoimmune diseases.

Antigen-specific B lymphocytes acquire proteoglycan aggrecan from cartilage extracellular matrix resulting in antigen presentation and CD4+ T-cell activation.

The majority of studies examining antigen-presenting cell (APC) function have focused on the capture and presentation of antigens released from pathogens or damaged cells. However, antigen-specific B cells are also capable of efficiently extracting antigens that are either tethered to, or integrally part of the plasma membrane of various target cells. In this study we show that B cells are also highly efficient at extracting integral components of the extracellular matrix (ECM) for subsequent presentation. In particular we demonstrate that B cells specific for aggrecan, an integral component of cartilage ECM, acquire this rheumatoid arthritis candidate autoantigen in both a B-cell-receptor-dependent and a contact-dependent manner. We also demonstrate that the subsequent presentation of aggregan from ECM leads to CD4(+) T-cell activation and effector cell formation. Recent studies have identified B-cell-mediated antigen presentation as essential for the development of autoimmunity, but a unique role for B cells compared with other APC has yet to be defined. Our findings lead us to propose that the acquisition of ECM-derived autoantigens represents a mechanism that defines the APC requirement for B cells in the development of autoimmunity.