Barbara Tolusso

Synovial tissue (ST) biopsy has evolved from an experimental procedure into a clinically relevant tool that provides unparalleled access to the inflamed joint in RA. Initially performed through blind needle and arthroscopic techniques, the adoption of minimally invasive ultrasound-guided biopsy has enabled safe, reproducible and patient-acceptable tissue sampling across large and small joints. Histopathological evaluation, including the Krenn Synovitis Score and recognition of distinct pathotypes, has provided diagnostic and prognostic insights, while comparative analyses have highlighted shared inflammatory pathways with OA and PsA. The integration of high-dimensional technologies such as bulk and single-cell transcriptomics, spatial profiling and proteomics has uncovered novel stromal and immune cell subsets and linked tissue signatures to therapeutic outcomes. Landmark biopsy-driven trials (R4RA and Synovial Tissue Research in Arthritis Pathobiology [STRAP]) have demonstrated the feasibility of stratifying treatment on the basis of synovial biology, positioning synovial biopsy as a cornerstone in the long journey towards precision RA management.

In a recent clinical trial, beetroot juice supplementation for 14 days yielded positive effects on systemic inflammation in adults with long COVID. Here, we explored the relationship between circulating markers of mitochondrial quality and inflammation in adults with long COVID as well as the impact of beetroot administration on those markers. We conducted secondary analyses of a placebo-controlled randomized clinical trial testing beetroot juice supplementation as a remedy against long COVID. Analyses were conducted in 25 participants, 10 assigned to placebo (mean age: 40.2 ± 11.5 years, 60% women) and 15 allocated to beetroot juice (mean age: 38.3 ± 7.7 years, 53.3% women). Extracellular vesicles were purified from serum by ultracentrifugation and assayed for components of the electron transport chain and mitochondrial DNA (mtDNA) by Western blot and droplet digital polymerase chain reaction (ddPCR), respectively. Inflammatory markers and circulating cell-free mtDNA were quantified in serum through a multiplex immunoassay and ddPCR, respectively. Beetroot juice administration for 14 days decreased serum levels of interleukin (IL)-1β, IL-8, and tumor necrosis factor alpha, with no effects on circulating markers of mitochondrial quality control. Significant negative associations were observed between vesicular markers of mitochondrial quality control and the performance on the 6 min walk test and flow-mediated dilation irrespective of group allocation. These findings suggest that an amelioration of mitochondrial quality, possibly mediated by mitochondria-derived vesicle recycling, may be among the mechanisms supporting improvements in physical performance and endothelial function during the resolution of long COVID.

Background/Objectives: Juvenile idiopathic arthritis (JIA) is a chronic childhood disease that often persists into the reproductive years. JIA may impact long-term fertility due to the prolonged exposure to immunosuppressive therapies. Methods: A total of 35 adult JIA female patients of childbearing age and 20 age-matched healthy controls were studied to test their anti-Müllerian hormone (AMH) serum levels as a biomarker of ovarian reserve. Demographic characteristics, disease duration, previous and current treatments, disease activity (DAS44), and a health assessment questionnaire (HAQ) were recorded. Results: JIA patients had a mean age of 22.3 ± 2.9 years, a disease duration of 12.3 ± 6.1 years, and a DAS44 of 1.24 ± 0.61. No differences were found in AMH serum levels between JIA and controls (5.78 ± 2.37 ng/mL vs. 6.60 ± 2.68 ng/mL, respectively; p = 0.17). Among the patients, 22 (62.9%) were receiving a stable dose of methotrexate (MTX) and 19 (54.3%) a dose of TNFα inhibitors. No difference in AMH serum levels was observed between JIA patients who were or were not exposed to MTX (p = 0.29) or to TNFα inhibitors (p = 0.50). Conclusions: Ovarian reserve as assessed by AMH serum levels appears to be comparable between those with JIA and age-matched controls and does not appear to be influenced by disease characteristics or prior/concomitant exposure to immunosuppressive drugs.

Summary: Current rheumatoid arthritis (RA) treatments do not restore immune tolerance. Investigating dendritic cell (DC) populations in human synovial tissue (ST) may reveal pathways to re-instate tolerance in RA. With single-cell and spatial-transcriptomics of synovial tissue biopsies, validated by micro co-culture systems, we identified condition and niche-specific myeloid DC clusters with distinct differentiation trajectories and functions. Healthy synovium contains a unique tolerogenic AXL ^pos DC2 cluster in the superficial sublining layer. In active RA, a macrophage-rich lining-layer niche becomes populated with inflammatory DC3 clusters that specifically activate memory CCL5 ^pos TEM and CCL5 ^pos CXCL13 ^pos TPH, promoting synovitis. In the sublining lymphoid niche, CCR7 ^pos DC2 mReg specifically interact with naïve-T-cells, potentially driving the local expansion of new effector T-cells. Sustained remission sees the resolution of these niches but lacks the recovery of tolerogenic AXL ^pos DC2, indicating latent potential for disease flare. A human RA disease-flare model showed that the activation of blood predecessor of ST-DC3 clusters precedes the onset of inflammation in joints. Therapeutic strategies targeting pathogenic ST-DC3 clusters, or reinstating tolerogenic AXL ^pos DC2, may restore immune homeostasis in RA. In brief: Deconstruction of human RA synovium, using single-cell spatial transcriptomics and micro-culture systems, reveals distinct neighbourhoods within the synovial architecture across health, and RA patients with active disease or sustained remission. Discrete niches are identified that contain distinct myeloid DC clusters that differ in frequency, differentiation trajectories, and effector functions. Highlights: Human RA synovium exhibits condition and niche specific myeloid DC clusters that vary in their tissue differentiation trajectories and functions. ST-CD14 ^pos DC3 (iDC3) support inflammatory CCL5 ^pos TEM and CCL5 ^pos TPH cell activation in the hyperplastic lining layer. ST-CCR7 ^pos DC2 (mReg), driven by MIR155, interact with naïve-T-cells in sublining lymphoid niches. A specific inflammatory signature of blood predecessors of ST-DC3s predict flare in RA. Graphical

Primary Sjögren’s disease is primarily driven by B-cell activation and is associated with a high risk of developing non-Hodgkin’s lymphoma (NHL). Over the last few decades, microRNA-155 (miR-155) has arisen as a key regulator of B-cells. Nevertheless, its role in primary Sjögren’s disease remains elusive. Thus, the purpose of this study was (i) to explore miR-155, B-cell activating factor (BAFF)-receptor (BAFF-R), and Interleukin 6 receptor (IL-6R) expression in the labial salivary glands (LSG) of patients with primary Sjögren’s disease, aiming to identify potential B-cell activation biomarkers related to NHL development. Twenty-four patients with primary Sjögren’s disease, and with available tissue blocks from a LSG biopsy performed at diagnosis, were enrolled. Among them, five patients developed B-cell NHL during follow-up (7.3 ± 3.1 years). A comparison group of 20 individuals with sicca disease was included. Clinical and laboratory parameters were recorded and the LSG biopsies were evaluated to assess local inflammation in terms of miR-155/BAFF-R and IL-6R expression. Stratifying the primary Sjögren’s disease cohort according to lymphomagenesis, miR-155 was upregulated in primary Sjögren’s disease patients who experienced NHL, more so than those who did not experience NHL. Moreover, miR-155 expression correlated with the focus score (FS), as well as BAFF-R and IL-6R expression, which were increased in primary Sjögren’s disease patients and in turn related to neoplastic evolution. In conclusion, epigenetic modulation may play a crucial role in the aberrant activation of B-cells in primary Sjögren’s disease, profoundly impacting the risk of NHL development.

Introduction: If properly evaluated, chronic kidney disease can be found in up to 50% of patients with systemic sclerosis (SSc). The renal resistive index (RRI) is a marker of intrarenal vascular resistance and can predict SSc-associated vasculopathy. This study aimed to determine the impact of bosentan, a nonselective endothelin-1 receptor antagonist, on RRI and kidney function in SSc patients with recurrent digital ulcers. Methods: Twenty-one patients (age 57 ± 9 years, 19 females) were recruited in a 16-week prospective open-label uncontrolled study. Standardized procedures were used to measure general clinical and laboratory characteristics, systolic, diastolic, and mean arterial pressure (MAP), pulse pressure (PP), diastolic to systolic blood pressure (D/S) ratio, and urinary endothelin-1 levels. The Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation was used to calculate kidney function as an estimated glomerular filtration rate (eGFR). RRI was measured by Doppler ultrasound as the average of three samplings of intrarenal blood flow in different kidney regions of both kidneys. Patients with secondary causes of kidney disease or kidney diseases associated with albuminuria were excluded. Results: Bosentan treatment for 16 weeks did not change RRI (0.731 ± 0.049–0.730 ± 0.054, p = 0.925), but increased urine endothelin-1 to creatinine ratio (0.27 ± 0.15–0.49 ± 0.57 pg/mg, p = 0.032) and reduced MAP (123 ± 10–101 ± 11 mm Hg, p < 0.001), PP (76 ± 11–68 ± 10 mm Hg, p = 0.003), D/S ratio (0.563 ± 0.044–0.538 ± 0.031, p = 0.006), and eGFR (92 ± 20–84 ± 24 mL/min/1.73 m2, p = 0.003). Discussion/Conclusion: In conclusion, in patients with SSc complicated by digital ulcers and normal to mildly diminished kidney function, bosentan had no effect on intrarenal hemodynamics, but reduced blood pressure levels and kidney function.

Background Knee Osteoarthritis (KOA) is one of the most frequent causes of pain and disability, representing a relevant clinical burden. Although it was largely considered a mostly mechanical-based pathology, low-grade synovial inflammation was found to contribute to radiographic progression and pain chronicity ^[1]. Objectives To evaluate the histological characteristics of synovial membrane inflammation in KOA patients compared to chronic inflammatory diseases as Rheumatoid (RA) and Psoriatic Arthritis (PsA) across disease phases. Methods 170 patients with KOA, 240 patients with RA, 92 patients with PsA naive to pharmacological treatments and 200 patients with RA in sustained clinical and ultrasound remission (RA-r) underwent ultrasound-guided synovial biopsy of the knee and conventional X-rays. None of enrolled KOA patient had evidence of inflammatory condition (including chronic inflammatory diseases and crystal related arthritis). Synovial membrane samples were processed for histology (hematoxylin-eosin staining) for the semiquantitative assessment of synovitis degree (Krenn synovitis score ^[2] - KSS). Moreover, this analysis was implemented with the semiqualitative assessment of presence/absence of synovial lymphocytes, plasmacells, myxoid degeneration and inflammatory perivascular aggregates. X-rays were scored for Kellgren-Lawrence score ^[3] (KLs) assessment. Results RA and PsA patients naive to pharmacological treatment showed higher degree of synovial inflammation than patients with KOA as well as more likely presence of lymphocytes and plasmacells aggregates. Considering the whole KOA population, total KSS directly correlated with age (R²=0.041; p=0.008), thickness of synovial membrane (R²=0.115; pConclusion KOA-related synovitis represents an unmet clinical need due to still uncovered pathogenetic mechanisms. The severity of KOA-related synovitis impacts on bone structural damage and is related to age, supporting the role of inflammageing in KOA pathogenesis. References [1]Sanchez-Lopez, E., Coras, R., Torres, A., Lane, N. E. & Guma, M. Synovial inflammation in osteoarthritis progression. Nature Reviews Rheumatology vol. 18 Preprint at https://doi.org/10.1038/s41584-022-00749-9 (2022). [2]Krenn, V. et al. Synovitis score: Discrimination between chronic low-grade and high-grade synovitis. Histopathology 49, (2006). [3]KELLGREN, J. H. & LAWRENCE, J. S. Radiological assessment of osteo-arthrosis. Ann Rheum Dis 16, (1957). Acknowledgements: NIL. Disclosure of Interests None Declared.

Background Subclinical tenosynovitis arose as a primary sign of inflammation already in the pre-clinical phases of Rheumatoid Arthritis (RA) and tenosynovitis is a common feature of active state and might persist although sustained remission is achieved. Objectives To assess the histological features of (teno)synovium from the extensor digitorum tendons of the hand across RA trajectory and to compare it with paired synovial tissue samples. Methods Thirty-seven patients underwent minimally invasive ultrasound (US)-guided biopsy of the tenosynovium of extensor digitorum tendons [n=16 with clinically suspected arthralgia (CSA), n=19 with RA (n=4 naïve, n=6 resistant to csDMARDS, n=8 to bDMARDs, and n=1 in sustained remission respectively)]. Twenty-three out of 37 patients presenting CSA or clinically evident synovitis in a peripheral joint (whether anatomically adjacent or not to the tendon of interest) underwent US-guided synovial tissue (ST) biopsy also in that location. The Krenn synovitis score (KSS) and the inflammatory infiltrate using CD68/CD21/CD3/CD20 immunohistochemical (IHC) analysis were assessed for each sample. Results Tenosynovium belonging to the extensor digitorum compartment of the wrist was successfully collected using a minimally invasive US-guided technique that enabled to retrieve representative samples in terms of lining and sublining layers presence in 86.5% and 100% of cases, respectively. Considering the pre-arthritis subgroup, KSS was similar in ACPA/RF^pos vs ACPA/RF^neg CSA (p=0.5814) and KSS was contingent with RA activity, being significantly higher in naïve or resistant RA (ANOVA p0.999) and IHC showed comparable enrichment in terms of lining and sublining CD68^pos, sublining CD20^pos and CD3^pos cells (for all comparisons p>0.05) between the two sites, in naive and c- or b-DMARDs resistant RA patients. The procedure was well tolerated, and no tendon injury occurred within 10 months follow-up. One patient developed local hematoma that recovered after topic treatment. Conclusion (Teno)synovium is a retrievable tissue through a minimally invasive procedure across RA trajectory and its inflammation is directly related to the disease state. Semiquantitative analysis showed comparable inflammatory degree and inflammatory cellular composition in adjacent anatomical sites of the same person. REFERENCES: NIL. Acknowledgements: NIL. Disclosure of Interests None Declared.