Molecular Determinants of Neutrophil Extracellular Vesicles That Drive Cartilage Regeneration in Inflammatory Arthritis.

OBJECTIVE: This study was undertaken to establish the potential therapeutic profile of neutrophil-derived extracellular vesicles (EVs) in experimental inflammatory arthritis and associate pharmacological activity with specific EV components, focusing on microRNAs. METHODS: Neutrophil EVs were administered intra-articularly through a prophylactic or therapeutic protocol to male C57BL/6 mice undergoing serum-transfer-induced inflammatory arthritis. Transcriptomic analysis of knees was performed on joints following EV administration, naive and arthritic mice (untreated; n = 4/group) and EV-treated diseased mice (intra-articular administration) with contralateral (vehicle-treated; n = 8/group). Comparison of healthy donor and patients with rheumatoid arthritis (RA) neutrophil EVs was performed. RESULTS: EVs afforded cartilage protection with an increase in collagen-II and reduced collagen-X expression within the joint. To gain mechanistic insights, RNA sequencing of the arthritic joints was conducted. A total of 5,231 genes were differentially expressed (P < 0.05), with 257 unique to EV treatment. EVs affected key regenerative pathways involved in joint development, including Wnt and Notch signaling. This wealth of genomic alteration prompted to identify microRNAs in EVs, 10 of which are associated with RA. As a proof of concept, we focused on miR-455-3p, which was detected in both healthy donor and RA EVs. EV addition to chondrocyte cultures elevated miR-455-3p and exerted anticatabolic effects upon interleukin-1ß stimulation; these effects were blocked by actinomycin or miR-455-3p antagomir. CONCLUSION: Neutrophils from patients with RA yielded EVs with composition, efficacy, and miR-455-3p content similar to those of healthy volunteers, suggesting that neutrophil EVs could be developed as an autologous treatment to protect and repair joint tissue of patients affected by inflammatory arthritides.

Could Gas6/TAM Axis Provide Valuable Insights into the Pathogenesis of Systemic Sclerosis?

Systemic sclerosis (SSc) is a connective tissue disorder characterized by microvascular injury, extracellular matrix deposition, autoimmunity, inflammation, and fibrosis. The clinical complexity and high heterogeneity of the disease make the discovery of potential therapeutic targets difficult. However, the recent progress in the comprehension of its pathogenesis is encouraging. Growth Arrest-Specific 6 (Gas6) and Tyro3, Axl, and MerTK (TAM) receptors are involved in multiple biological processes, including modulation of the immune response, phagocytosis, apoptosis, fibrosis, inflammation, cancer development, and autoimmune disorders. In the present manuscript, we review the current evidence regarding SSc pathogenesis and the role of the Gas6/TAM system in several human diseases, suggesting its likely contribution in SSc and highlighting areas where further research is necessary to fully comprehend the role of TAM receptors in this condition. Indeed, understanding the involvement of TAM receptors in SSc, which is currently unknown, could provide valuable insights for novel potential therapeutic targets.

Clinical Phenotypes, Serological Biomarkers, and Synovial Features Defining Seropositive and Seronegative Rheumatoid Arthritis: A Literature Review.

Rheumatoid arthritis (RA) is a chronic autoimmune disorder which can lead to long-term joint damage and significantly reduced quality of life if not promptly diagnosed and adequately treated. Despite significant advances in treatment, about 40% of patients with RA do not respond to individual pharmacological agents and up to 20% do not respond to any of the available medications. To address this large unmet clinical need, several recent studies have focussed on an in-depth histological and molecular characterisation of the synovial tissue to drive the application of precision medicine to RA. Currently, RA patients are clinically divided into "seropositive" or "seronegative" RA, depending on the presence of routinely checked antibodies. Recent work has suggested that over the last two decades, long-term outcomes have improved significantly in seropositive RA but not in seronegative RA. Here, we present up-to-date differences in epidemiology, clinical features, and serological biomarkers in seronegative versus seropositive RA and discuss how histological and molecular synovial signatures, revealed by recent large synovial biopsy-based clinical trials, may be exploited to refine the classification of RA patients, especially in the seronegative group.

Axl and MerTK regulate synovial inflammation and are modulated by IL-6 inhibition in rheumatoid arthritis.

The TAM tyrosine kinases, Axl and MerTK, play an important role in rheumatoid arthritis (RA). Here, using a unique synovial tissue bioresource of patients with RA matched for disease stage and treatment exposure, we assessed how Axl and MerTK relate to synovial histopathology and disease activity, and their topographical expression and longitudinal modulation by targeted treatments. We show that in treatment-naive patients, high AXL levels are associated with pauci-immune histology and low disease activity and inversely correlate with the expression levels of pro-inflammatory genes. We define the location of Axl/MerTK in rheumatoid synovium using immunohistochemistry/fluorescence and digital spatial profiling and show that Axl is preferentially expressed in the lining layer. Moreover, its ectodomain, released in the synovial fluid, is associated with synovial histopathology. We also show that Toll-like-receptor 4-stimulated synovial fibroblasts from patients with RA modulate MerTK shedding by macrophages. Lastly, Axl/MerTK synovial expression is influenced by disease stage and therapeutic intervention, notably by IL-6 inhibition. These findings suggest that Axl/MerTK are a dynamic axis modulated by synovial cellular features, disease stage and treatment.

Molecular portrait of chronic joint diseases: Defining endotypes toward personalized medicine.

Joint diseases affect hundreds of millions of people worldwide, and their prevalence is constantly increasing. To date, despite recent advances in the development of therapeutic options for most rheumatic conditions, a significant proportion of patients still lack efficient disease management, considerably impacting their quality of life. Through the spectrum of rheumatoid arthritis (RA), psoriatic arthritis (PsA), and osteoarthritis (OA) as quintessential and common rheumatic diseases, this review first provides an overview of their epidemiological and clinical features before exploring how the better definition of clinical phenotypes has helped their clinical management. It then discusses the recent progress in understanding the diversity of endotypes underlying disease phenotypes. Finally, this review highlights the current challenges of implementing molecular endotypes towards the personalized management of RA, PsA and OA patients in the future.

Comparative analysis of late-stage rheumatoid arthritis and osteoarthritis reveals shared histopathological features.

OBJECTIVES: Osteoarthritis (OA) is a debilitating and heterogeneous condition, characterized by various levels of articular cartilage degradation, osteophytes formation, and synovial inflammation. Multiple evidences suggest that synovitis may appear early in the disease development and correlates with disease severity and pain, therefore representing a relevant therapeutic target. In a typical synovitis-driven joint disease, namely rheumatoid arthritis (RA), several pathotypes have been described by our group and associated with clinical phenotypes, disease progression, and response to therapy. However, whether these pathotypes can be also observed in the OA synovium is currently unknown. METHODS: Here, using histological approaches combined with semi-quantitative scoring and quantitative digital image analyses, we comparatively characterize the immune cell infiltration in a large cohort of OA and RA synovial tissue samples collected at the time of total joint replacement. RESULTS: We demonstrate that OA synovium can be categorized also into three pathotypes and characterized by disease- and stage-specific features. Moreover, we revealed that pathotypes specifically reflect distinct levels of peripheral inflammation. CONCLUSIONS: In this study, we provide a novel and relevant pathological classification of OA synovial inflammation. Further studies investigating synovial molecular pathology in OA may contribute to the development of disease-modifying therapies.

Serum and Tissue Biomarkers Associated With Composite of Relevant Endpoints for Sjögren Syndrome (CRESS) and Sjögren Tool for Assessing Response (STAR) to B Cell-Targeted Therapy in the Trial of Anti-B Cell Therapy in Patients With Primary Sjögren Syndrome (TRACTISS).

OBJECTIVE: This study aimed to identify peripheral and salivary gland (SG) biomarkers of response/resistance to B cell depletion based on the novel concise Composite of Relevant Endpoints for Sjögren Syndrome (cCRESS) and candidate Sjögren Tool for Assessing Response (STAR) composite endpoints. METHODS: Longitudinal analysis of peripheral blood and SG biopsies was performed pre- and post-treatment from the Trial of Anti-B Cell Therapy in Patients With Primary Sjögren Syndrome (TRACTISS) combining flow cytometry immunophenotyping, serum cytokines, and SG bulk RNA sequencing. RESULTS: Rituximab treatment prevented the worsening of SG inflammation observed in the placebo arm, by inhibiting the accumulation of class-switched memory B cells within the SG. Furthermore, rituximab significantly down-regulated genes involved in immune-cell recruitment, lymphoid organization alongside antigen presentation, and T cell co-stimulatory pathways. In the peripheral compartment, rituximab down-regulated immunoglobulins  and auto-antibodies together with pro-inflammatory cytokines and chemokines. Interestingly, patients classified as responders  according to STAR displayed significantly higher baseline levels of C-X-C motif chemokine ligand-13 (CXCL13), interleukin (IL)-22, IL-17A, IL-17F, and tumor necrosis factor-α (TNF-α), whereas a longitudinal analysis of serum T cell-related cytokines showed a selective reduction in both STAR and cCRESS responder patients. Conversely, cCRESS response was better associated with biomarkers of SG immunopathology, with cCRESS-responders showing a significant decrease in SG B cell infiltration and reduced expression of transcriptional gene modules related to T cell costimulation, complement activation, and Fcγ-receptor engagement. Finally, cCRESS and STAR response were associated with a significant improvement in SG exocrine function linked to transcriptional evidence of SG epithelial and metabolic restoration. CONCLUSION: Rituximab modulates both peripheral and SG inflammation, preventing the deterioration of exocrine function with functional and metabolic restoration of the glandular epithelium. Response assessed by newly developed cCRESS and STAR criteria was associated with differential modulation of peripheral and SG biomarkers, emerging as novel tools for patient stratification.

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.

Stratification of biological therapies by pathobiology in biologic-naive patients with rheumatoid arthritis (STRAP and STRAP-EU): two parallel, open-label, biopsy-driven, randomised trials.

BACKGROUND: Despite highly effective targeted therapies for rheumatoid arthritis, about 40% of patients respond poorly, and predictive biomarkers for treatment choices are lacking. We did a biopsy-driven trial to compare the response to rituximab, etanercept, and tocilizumab in biologic-naive patients with rheumatoid arthritis stratified for synovial B cell status. METHODS: STRAP and STRAP-EU were two parallel, open-label, biopsy-driven, stratified, randomised, phase 3 trials done across 26 university centres in the UK and Europe. Biologic-naive patients aged 18 years or older with rheumatoid arthritis based on American College of Rheumatology (ACR)-European League Against Rheumatism classification criteria and an inadequate response to conventional synthetic disease-modifying antirheumatic drugs (DMARDs) were included. Following ultrasound-guided synovial biopsy, patients were classified as B cell poor or B cell rich according to synovial B cell signatures and randomly assigned (1:1:1) to intravenous rituximab (1000 mg at week 0 and week 2), subcutaneous tocilizumab (162 mg per week), or subcutaneous etanercept (50 mg per week). The primary outcome was the 16-week ACR20 response in the B cell-poor, intention-to-treat population (defined as all randomly assigned patients), with data pooled from the two trials, comparing etanercept and tocilizumab (grouped) versus rituximab. Safety was assessed in all patients who received at least one dose of study drug. These trials are registered with the EU Clinical Trials Register, 2014-003529-16 (STRAP) and 2017-004079-30 (STRAP-EU). FINDINGS: Between June 8, 2015, and July 4, 2019, 226 patients were randomly assigned to etanercept (n=73), tocilizumab (n=74), and rituximab (n=79). Three patients (one in each group) were excluded after randomisation because they received parenteral steroids in the 4 weeks before recruitment. 168 (75%) of 223 patients in the intention-to-treat population were women and 170 (76%) were White. In the B cell-poor population, ACR20 response at 16 weeks (primary endpoint) showed no significant differences between etanercept and tocilizumab grouped together and rituximab (46 [60%] of 77 patients vs 26 [59%] of 44; odds ratio 1·02 [95% CI 0·47-2·17], p=0·97). No differences were observed for adverse events, including serious adverse events, which occurred in six (6%) of 102 patients in the rituximab group, nine (6%) of 108 patients in the etanercept group, and three (4%) of 73 patients in the tocilizumab group (p=0·53). INTERPRETATION: In this biologic-naive population of patients with rheumatoid arthrtitis, the dichotomic classification into synovial B cell poor versus rich did not predict treatment response to B cell depletion with rituximab compared with alternative treatment strategies. However, the lack of response to rituximab in patients with a pauci-immune pathotype and the higher risk of structural damage progression in B cell-rich patients treated with rituximab warrant further investigations into the ability of synovial tissue analyses to inform disease pathogenesis and treatment response. FUNDING: UK Medical Research Council and Versus Arthritis.

Synovial Inflammatory Pathways Characterize Anti-TNF-Responsive Rheumatoid Arthritis Patients.

OBJECTIVE: This study was undertaken to understand the mechanistic basis of response to anti-tumor necrosis factor (anti-TNF) therapies and to determine whether transcriptomic changes in the synovium are reflected in peripheral protein markers. METHODS: Synovial tissue from 46 rheumatoid arthritis (RA) patients was profiled with RNA sequencing before and 12 weeks after treatment with anti-TNF therapies. Pathway and gene signature analyses were performed on RNA expression profiles of synovial biopsies to identify mechanisms that could discriminate among patients with a good response, a moderate response, or no response, according to the American College of Rheumatology (ACR)/EULAR response criteria. Serum proteins encoded by synovial genes that were differentially expressed between ACR/EULAR response groups were measured in the same patients. RESULTS: Gene signatures predicted which patients would have good responses, and pathway analysis identified elevated immune pathways, including chemokine signaling, Th1/Th2 cell differentiation, and Toll-like receptor signaling, uniquely in good responders. These inflammatory pathways were correspondingly down-modulated by anti-TNF therapy only in good responders. Based on cell signature analysis, lymphocyte, myeloid, and fibroblast cell populations were elevated in good responders relative to nonresponders, consistent with the increased inflammatory pathways. Cell signatures that decreased following anti-TNF treatment were predominately associated with lymphocytes, and fewer were associated with myeloid and fibroblast populations. Following anti-TNF treatment, and only in good responders, several peripheral inflammatory proteins decreased in a manner that was consistent with corresponding synovial gene changes. CONCLUSION: Collectively, these data suggest that RA patients with robust responses to anti-TNF therapies are characterized at baseline by immune pathway activation, which decreases following anti-TNF treatment. Understanding mechanisms that define patient responsiveness to anti-TNF treatment may assist in development of predictive markers of patient response and earlier treatment options.

Rituximab versus tocilizumab in rheumatoid arthritis: synovial biopsy-based biomarker analysis of the phase 4 R4RA randomized trial.

Patients with rheumatoid arthritis (RA) receive highly targeted biologic therapies without previous knowledge of target expression levels in the diseased tissue. Approximately 40% of patients do not respond to individual biologic therapies and 5-20% are refractory to all. In a biopsy-based, precision-medicine, randomized clinical trial in RA (R4RA; n = 164), patients with low/absent synovial B cell molecular signature had a lower response to rituximab (anti-CD20 monoclonal antibody) compared with that to tocilizumab (anti-IL6R monoclonal antibody) although the exact mechanisms of response/nonresponse remain to be established. Here, in-depth histological/molecular analyses of R4RA synovial biopsies identify humoral immune response gene signatures associated with response to rituximab and tocilizumab, and a stromal/fibroblast signature in patients refractory to all medications. Post-treatment changes in synovial gene expression and cell infiltration highlighted divergent effects of rituximab and tocilizumab relating to differing response/nonresponse mechanisms. Using ten-by-tenfold nested cross-validation, we developed machine learning algorithms predictive of response to rituximab (area under the curve (AUC) = 0.74), tocilizumab (AUC = 0.68) and, notably, multidrug resistance (AUC = 0.69). This study supports the notion that disease endotypes, driven by diverse molecular pathology pathways in the diseased tissue, determine diverse clinical and treatment-response phenotypes. It also highlights the importance of integration of molecular pathology signatures into clinical algorithms to optimize the future use of existing medications and inform the development of new drugs for refractory patients.

Circulating and Synovial Pentraxin-3 (PTX3) Expression Levels Correlate With Rheumatoid Arthritis Severity and Tissue Infiltration Independently of Conventional Treatments Response.

Aims: To determine the relationship between PTX3 systemic and synovial levels and the clinical features of rheumatoid arthritis (RA) in a cohort of early, treatment naïve patients and to explore the relevance of PTX3 expression in predicting response to conventional-synthetic (cs) Disease-Modifying-Anti-Rheumatic-Drugs (DMARDs) treatment. Methods: PTX3 expression was analyzed in 119 baseline serum samples from early naïve RA patients, 95 paired samples obtained 6-months following the initiation of cs-DMARDs treatment and 43 healthy donors. RNA-sequencing analysis and immunohistochemistry for PTX3 were performed on a subpopulation of 79 and 58 synovial samples, respectively, to assess PTX3 gene and protein expression. Immunofluorescence staining was performed to characterize PTX3 expressing cells within the synovium. Results: Circulating levels of PTX3 were significantly higher in early RA compared to healthy donors and correlated with disease activity at baseline and with the degree of structural damages at 12-months. Six-months after commencing cs-DMARDs, a high level of PTX3, proportional to the baseline value, was still detectable in the serum of patients, regardless of their response status. RNA-seq analysis confirmed that synovial transcript levels of PTX3 correlated with disease activity and the presence of mediators of inflammation, tissue remodeling and bone destruction at baseline. PTX3 expression in the synovium was strongly linked to the degree of immune cell infiltration, the presence of ectopic lymphoid structures and seropositivity for autoantibodies. Accordingly, PTX3 was found to be expressed by numerous synovial cell types such as plasma cells, fibroblasts, vascular and lymphatic endothelial cells, macrophages, and neutrophils. The percentage of PTX3-positive synovial cells, although significantly reduced at 6-months post-treatment as a result of global decreased cellularity, was similar in cs-DMARDs responders and non-responders. Conclusion: This study demonstrates that, early in the disease and prior to treatment modification, the level of circulating PTX3 is a reliable marker of RA activity and predicts a high degree of structural damages at 12-months. In the joint, PTX3 associates with immune cell infiltration and the presence of ectopic lymphoid structures. High synovial and peripheral blood levels of PTX3 are associated with chronic inflammation characteristic of RA. Additional studies to determine the mechanistic link are required.

PD-L1 signaling on human memory CD4+ T cells induces a regulatory phenotype.

Programmed cell death protein 1 (PD-1) is expressed on T cells upon T cell receptor (TCR) stimulation. PD-1 ligand 1 (PD-L1) is expressed in most tumor environments, and its binding to PD-1 on T cells drives them to apoptosis or into a regulatory phenotype. The fact that PD-L1 itself is also expressed on T cells upon activation has been largely neglected. Here, we demonstrate that PD-L1 ligation on human CD25-depleted CD4+ T cells, combined with CD3/TCR stimulation, induces their conversion into highly suppressive T cells. Furthermore, this effect was most prominent in memory (CD45RA-CD45RO+) T cells. PD-L1 engagement on T cells resulted in reduced ERK phosphorylation and decreased AKT/mTOR/S6 signaling. Importantly, T cells from rheumatoid arthritis patients exhibited high basal levels of phosphorylated ERK and following PD-L1 cross-linking both ERK signaling and the AKT/mTOR/S6 pathway failed to be down modulated, making them refractory to the acquisition of a regulatory phenotype. Altogether, our results suggest that PD-L1 signaling on memory T cells could play an important role in resolving inflammatory responses; maintaining a tolerogenic environment and its failure could contribute to ongoing autoimmunity.

Novel Bispecific Antibody for Synovial-Specific Target Delivery of Anti-TNF Therapy in Rheumatoid Arthritis.

Biologic drugs, especially anti-TNF, are considered as the gold standard therapy in rheumatoid arthritis. However, non-uniform efficacy, incidence of infections, and high costs are major concerns. Novel tissue-specific agents may overcome the current limitations of systemic administration, providing improved potency, and safety. We developed a bispecific antibody (BsAb), combining human arthritic joint targeting, via the synovial-specific single-chain variable fragment (scFv)-A7 antibody, and TNFα neutralization, via the scFv-anti-TNFα of adalimumab, with the binding/blocking capacity comparable to adalimumab -immunoglobulin G (IgG). Tissue-targeting capacity of the BsAb was confirmed on the human arthritic synovium in vitro and in a synovium xenograft Severe combined immune deficient (SCID) mouse model. Peak graft accumulation occurred at 48 h after injection with sustained levels over adalimumab-IgG for 7 days and increased therapeutic effect, efficiently decreasing tissue cellularity, and markers of inflammation with higher potency compared to the standard treatment. This study provides the first description of a BsAb capable of drug delivery, specifically to the disease tissue, and a strong evidence of improved therapeutic effect on the human arthritic synovium, with applications to other existing biologics.

Rituximab versus tocilizumab in anti-TNF inadequate responder patients with rheumatoid arthritis (R4RA): 16-week outcomes of a stratified, biopsy-driven, multicentre, open-label, phase 4 randomised controlled trial.

BACKGROUND: Although targeted biological treatments have transformed the outlook for patients with rheumatoid arthritis, 40% of patients show poor clinical response, which is mechanistically still unexplained. Because more than 50% of patients with rheumatoid arthritis have low or absent CD20 B cells-the target for rituximab-in the main disease tissue (joint synovium), we hypothesised that, in these patients, the IL-6 receptor inhibitor tocilizumab would be more effective. The aim of this trial was to compare the effect of tocilizumab with rituximab in patients with rheumatoid arthritis who had an inadequate response to anti-tumour necrosis factor (TNF) stratified for synovial B-cell status. METHODS: This study was a 48-week, biopsy-driven, multicentre, open-label, phase 4 randomised controlled trial (rituximab vs tocilizumab in anti-TNF inadequate responder patients with rheumatoid arthritis; R4RA) done in 19 centres across five European countries (the UK, Belgium, Italy, Portugal, and Spain). Patients aged 18 years or older who fulfilled the 2010 American College of Rheumatology and European League Against Rheumatism classification criteria for rheumatoid arthritis and were eligible for treatment with rituximab therapy according to UK National Institute for Health and Care Excellence guidelines were eligible for inclusion in the trial. To inform balanced stratification, following a baseline synovial biopsy, patients were classified histologically as B-cell poor or rich. Patients were then randomly assigned (1:1) centrally in block sizes of six and four to receive two 1000 mg rituximab infusions at an interval of 2 weeks (rituximab group) or 8 mg/kg tocilizumab infusions at 4-week intervals (tocilizumab group). To enhance the accuracy of the stratification of B-cell poor and B-cell rich patients, baseline synovial biopsies from all participants were subjected to RNA sequencing and reclassified by B-cell molecular signature. The study was powered to test the superiority of tocilizumab over rituximab in the B-cell poor population at 16 weeks. The primary endpoint was defined as a 50% improvement in Clinical Disease Activity Index (CDAI50%) from baseline. The trial is registered on the ISRCTN database, ISRCTN97443826, and EudraCT, 2012-002535-28. FINDINGS: Between Feb 28, 2013, and Jan 17, 2019, 164 patients were classified histologically and were randomly assigned to the rituximab group (83 [51%]) or the tocilizumab group (81 [49%]). In patients histologically classified as B-cell poor, there was no statistically significant difference in CDAI50% between the rituximab group (17 [45%] of 38 patients) and the tocilizumab group (23 [56%] of 41 patients; difference 11% [95% CI -11 to 33], p=0·31). However, in the synovial biopsies classified as B-cell poor with RNA sequencing the tocilizumab group had a significantly higher response rate compared with the rituximab group for CDAI50% (rituximab group 12 [36%] of 33 patients vs tocilizumab group 20 [63%] of 32 patients; difference 26% [2 to 50], p=0·035). Occurrence of adverse events (rituximab group 76 [70%] of 108 patients vs tocilizumab group 94 [80%] of 117 patients; difference 10% [-1 to 21) and serious adverse events (rituximab group 8 [7%] of 108 vs tocilizumab group 12 [10%] of 117; difference 3% [-5 to 10]) were not significantly different between treatment groups. INTERPRETATION: The results suggest that RNA sequencing-based stratification of rheumatoid arthritis synovial tissue showed stronger associations with clinical responses compared with histopathological classification. Additionally, for patients with low or absent B-cell lineage expression signature in synovial tissue tocilizumab is more effective than rituximab. Replication of the results and validation of the RNA sequencing-based classification in independent cohorts is required before making treatment recommendations for clinical practice. FUNDING: Efficacy and Mechanism Evaluation programme from the UK National Institute for Health Research.

Novel insights into macrophage diversity in rheumatoid arthritis synovium.

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease affecting joints and causing progressive damage and disability. Macrophages are of critical importance in the initiation and perpetuation of synovitis in RA, they can function as antigen presenting cells leading to T-cell dependent B-cell activation, assume a variety of inflammatory cell states with the production of destructive cytokines, but also contribute to tissue homeostasis/repair. The recent development of high-throughput technologies, including bulk and single cells RNA-sequencing, has broadened our understanding of synovial cell diversity, and opened novel perspectives to the discovery of new potential therapeutic targets in RA. In this review, we will focus on the relationship between the synovial macrophage infiltration and clinical disease severity and response to treatment. We will then provide a state-of-the-art picture of the biological roles of synovial macrophages and distinct macrophage subsets described in RA. Finally, we will review the effects of approved conventional and biologic drugs on the synovial macrophage component and highlight the therapeutic potential of future strategies to re-program macrophage phenotypes in RA.

Increased plasma levels of Gas6 and its soluble tyrosine kinase receptors Mer and Axl are associated with immunological activity and severity of lupus nephritis.

OBJECTIVES: Growth arrest-specific 6 (Gas6) and its receptors have been shown to play a crucial role in the homeostasis of the innate immune system by regulating apoptosis and inflammation. We aimed to verify whether an impairment of this system is associated with systemic lupus erythematosus (SLE) disease activity and with lupus nephritis (LN). METHODS: Plasma Gas6 and the soluble cleaved form of the receptors MerTK (sMer) and Axl (sAxl) concentrations were measured in n=59 SLE patients (n=44 with nephritis, 75%) and analysed in relationship to clinical and laboratory data. RESULTS: Patients with LN were characterised by higher Gas6 (19.0 ng/mL [16.8-24.5] vs. 16.5 ng/mL [13.89-18.91]; p=0.03) and sAxl plasma levels than those without LN (31.36 ng/mL [25.1-41.4] vs. 20.2 ng/mL [15.6-30.7]; p=0.03); conversely sMer plasma concentrations were similar between groups. All the three biomarkers studied were directly correlated to creatinine and daily proteinuria, being inversely related to creatinine clearance. 39 patients had a proteinuria level of <0.5 mg/day, 14 between 0.5 and 3.5 mg/day and 5 had ≥3.5 g/day; Gas6, sAxl and sMer plasma concentrations significantly increased for increasing degree of proteinuria (test for trend p=0.0002; p=0.02; p=0.009, respectively).These correlations were confirmed in multiple linear regression analysis models accounting for gender, age, disease duration and concomitant treatment. CONCLUSIONS: Plasma Gas6, sAxl and sMer concentrations are associated with the severity of LN in patients affected by SLE. The excess cleavage of TAM receptors might contribute to LN pathogenesis.

IL-23 skin and joint profiling in psoriatic arthritis: novel perspectives in understanding clinical responses to IL-23 inhibitors.

OBJECTIVES: To determine the relationship between synovial versus skin transcriptional/histological profiles in patients with active psoriatic arthritis (PsA) and explore mechanistic links between diseased tissue pathology and clinical outcomes. METHODS: Twenty-seven active PsA patients were enrolled in an observational/open-label study and underwent biopsies of synovium and paired lesional/non-lesional skin before starting anti-tumour necrosis factor (TNF) (if biologic-naïve) or ustekinumab (if anti-TNF inadequate responders). Molecular analysis of 80-inflammation-related genes and protein levels for interleukin (IL)-23p40/IL-23p19/IL-23R were assessed by real-time-PCR and immunohistochemistry, respectively. RESULTS: At baseline, all patients had persistent active disease as per inclusion criteria. At primary end-point (16-weeks post-treatment), skin responses favoured ustekinumab, while joint responses favoured anti-TNF therapies. Principal component analysis revealed distinct clustering of synovial tissue gene expression away from the matched skin. While IL12B, IL23A and IL23R were homogeneously expressed in lesional skin, their expression was extremely heterogeneous in paired synovial tissues. Here, IL-23 transcriptomic/protein expression was strongly linked to patients with high-grade synovitis who, however, were not distinguishable by conventional clinimetric measures. CONCLUSIONS: PsA synovial tissue shows a heterogeneous IL-23 axis profile when compared with matched skin. Synovial molecular pathology may help to identify among clinically indistinguishable patients those with a greater probability of responding to IL-23 inhibitors.

Distinct synovial tissue macrophage subsets regulate inflammation and remission in rheumatoid arthritis.

Immune-regulatory mechanisms of drug-free remission in rheumatoid arthritis (RA) are unknown. We hypothesized that synovial tissue macrophages (STM), which persist in remission, contribute to joint homeostasis. We used single-cell transcriptomics to profile 32,000 STMs and identified phenotypic changes in patients with early/active RA, treatment-refractory/active RA and RA in sustained remission. Each clinical state was characterized by different frequencies of nine discrete phenotypic clusters within four distinct STM subpopulations with diverse homeostatic, regulatory and inflammatory functions. This cellular atlas, combined with deep-phenotypic, spatial and functional analyses of synovial biopsy fluorescent activated cell sorted STMs, revealed two STM subpopulations (MerTKposTREM2high and MerTKposLYVE1pos) with unique remission transcriptomic signatures enriched in negative regulators of inflammation. These STMs were potent producers of inflammation-resolving lipid mediators and induced the repair response of synovial fibroblasts in vitro. A low proportion of MerTKpos STMs in remission was associated with increased risk of disease flare after treatment cessation. Therapeutic modulation of MerTKpos STM subpopulations could therefore be a potential treatment strategy for RA.