Osteoarthritis (OA) is a destructive joint disease affecting multiple tissues, including synovium. Previous studies have identified some distinct fibroblast subtypes within synovium; however, the characterization of fibroblast subsets during distinct stages of knee (K)OA disease, and their contributions to the endogenous mechanisms that drive synovial fibrosis during KOA, are not well characterized. Here we profile synovium from early- (KL I) and advanced- (KL III/IV) stages of radiographic KOA. First, bulk-RNA sequencing of early- and advanced-staged KOA synovial tissue revealed transcriptomic differences between the two disease stages. Using single-nuclei RNA sequencing (snRNA-seq) and flow cytometry, we identified distinct fibroblast subsets and uncovered an endotypic shift in fibroblast subsets during KOA pathogenesis, transitioning from DPP4+ in early-stage to ITGB8+ in advanced-stages. SnRNA-seq of synovium from mice with experimental KOA revealed analogous populations of Dpp4+ and Itgb8+ fibroblasts in tissue from early and advanced model stages. Human advanced-stage KOA synovial tissue had stronger expression of matrisome-annotated genes compared to early-stage tissue. BHLHE40, a crucial transcriptional regulator of ECM related genes, was identified as upregulated in ITGB8+ fibroblasts compared to DPP4+ fibroblasts. Using primary human OA fibroblasts in vitro, and conditional knock out mice in vivo, we found that fibroblast-intrinsic loss of BHLHE40 increased fibrosis-related gene expression, enhanced fibroblast activation and induced severe synovial fibrosis in vivo. In contrast, overexpression of BHLHE40 in vitro was able to suppress TGF-β-induced fibroblast activation. Overall, this study provides a comprehensive cellular atlas of KOA synovium and has identified BHLHE40 as a crucial regulator of fibroblast-mediated synovial fibrosis. ### Competing Interest Statement DE, EG, KT, JSR and MK have filed a provisional patent application on means and methods for the treatment of musculoskeletal diseases (#EP 25159912.2 and #EP 25159917.1).

Metabolic mediators play an important role in regulating chronic inflammation in the body. Here we report an unexpected role for GDF15 (Growth Differentiation Factor 15), a central mediator of food intake, in inflammation-associated bone loss. GDF15 serum levels were found to be elevated in arthritis patients and inversely correlated with bone density. Despite being associated with inflammation, we found that GDF15 itself does not cause, nor contribute to, clinical or histopathological arthritis. Rather, under inflammatory conditions, GDF15 mediates trabecular bone loss through its receptor GFRAL, which is exclusively expressed in the hindbrain. GDF15-GFRAL binding results in β-adrenergic activation of MALPs (Marrow Adipocytic Lineage Precursors) in the bone marrow, which stimulate osteoclasts and trigger bone loss. These data suggest a metabolic mediator-controlled brain-bone axis in inflammation, through which bone loss is induced in a contextual rather than general manner. These findings may lead to more specific therapeutic interventions to protect bone. FWO Immune Mechanisms of Human Disease (HUM)

Disclosure: S. Calvo Blanco: None. O. Nobus: None. T. Liekens: None. L. Verlinden: None. I. Stockmans: None. K. Moermans: None. F. Stappers: None. L. Crombez: None. C. Vercruysse: None. K. Van Belleghem: None. S. Lamon: None. E. Gracey: None. D. Elewaut: None. A. De Spiegeleer: None. R.I. Dmitriev: None. G.G. T'sjoen: None. L. Devisscher: None. S. Stegen: None. V. Dubois: None. BACKGROUND: Gender-affirming therapy for transgender adolescents often includes puberty suppression with gonadotropin-releasing hormone analogues (GnRHa), followed by gender-affirming hormones. In those assigned male at birth, the addition of a progestogen – such as natural progesterone (P4) or synthetic medroxyprogesterone acetate (MPA) – to the standard estrogen therapy has gained popularity due to a presumed enhanced feminization. However, the effects of progestogen supplementation on the musculoskeletal system and metabolic tissues remain unclear. To address this gap, we examined the impact of combined estrogen and progestogen therapy on various organ systems using a mouse model that replicates the clinical trajectory of transgender girls. METHODS: Prepubertal 4-week-old male mice received the GnRHa degarelix (DGX) to suppress puberty, followed by estradiol (E2) combined with a progestogen (P4 or MPA) from 8 weeks of age. Their phenotype at 16 weeks of age was compared to puberty-suppressed male mice treated with E2 alone, and to vehicle-treated male and female animals (controls for native sex and gender, respectively). To investigate metabolic changes, body composition was determined by MRI, fasting glucose and insulin levels were measured, and a glucose tolerance test was performed. The impact of the hormonal manipulations on muscle strength and fatigue were assessed by grip strength and treadmill endurance tests, and fiber type composition was determined by immunohistochemistry. Effects on the skeleton were investigated through microcomputed tomography, three-point bending and (dynamic) histomorphometry. RESULTS: DGX strongly decreased the weight of reproductive tissues including testes and seminal vesicles, confirming effective puberty suppression. E2 alone did not further affect reproductive tissue mass in DGX-treated animals, while E2 and progestogen co-administration increased testicular weight, albeit well-below control male levels. Puberty suppression shifted body composition towards increased fat mass and decreased lean mass, and E2 with or without progestogen did not fully reverse these changes. Fasting glucose levels were increased in mice treated with P4 or MPA without differences in glucose tolerance. Muscle strength was slightly higher in P4-treated mice. Resistance to fatigue was decreased upon puberty suppression and restored by E2, and this was accompanied by changes in fiber type composition. Bone strength was diminished by DGX, and restored to control levels by E2 without additional effect of P4 or MPA. CONCLUSION: Addition of a progestogen does not profoundly modulate the effect of estradiol on the body composition and the musculoskeletal system but increases fasting glucose levels in this mouse model of adolescent gender transition. Presentation: Sunday, July 13, 2025

Background: Osteoarthritis (OA) is a degenerative joint disease affecting multiple tissues in the joint including the synovium. The synovium is a connective tissue ensuring joint lubrication however, in OA, the synovium undergoes substantial changes including inflammation, hyperplasia and cellular proliferation. This study sought to identify key cellular components involved in OA synovial pathology utilizing single-nuclei RNA sequencing (snRNA). Objectives: 1. Identify cell types and distinct subtypes in the synovium of early (KL1) and late (KL3/4) stage radiographic knee OA and their associated transcriptomic signatures using snRNAseq. 2. Determine the presence of identified human OA synovium cell subtypes in the synovium of destabilization of the medial meniscus (DMM)-OA mice using snRNAseq of the murine synovium. 3. Investigate the contribution of key transcriptional regulators in ECM/fibrosis regulatory mechanisms in vitro and in vivo. Methods: SnRNA sequencing was performed on human OA knee synovial tissues from early- (KL1; n=5) and late- (KL3/4; n=4) stage radiographic knee OA patients and mouse synovia from naïve as control (n=3), 2 weeks (n=4) and 10 weeks (n=3) post destabilization of the medial meniscus (DMM) surgery. Bulk RNA sequencing was performed on human OA synovium from patients with KL1 (n=6) and KL3/4 (n=8) radiographic knee OA. Flow cytometry was performed on cryopreserved synovium from healthy donors (n=5), early (n=10) and late (n=14). Bioinformatics analysis was performed to discern cell types and subtypes using canonical markers, differentially expressed genes (DEG), key pathways, and putative transcriptional regulators implicated in disease progression. Results: SnRNA sequencing of 25285 nuclei from human knee synovial tissue identified fibroblasts to be the predominant cell type, comprising at least 50% of the cellular composition from KL1 and KL3/4 graded radiographic knee OA synovium. Clustering analysis of fibroblasts resolved nine fibroblast subtypes. An endophenotypic shift in fibroblast subsets was noted with disease stage. Synovia from early-stage OA contributed a higher proportion of nuclei to subclusters 1, 2, 4 and 6 while late-stage synovium predominantly contributed nuclei to subclusters 0, 3 and 5 (FIGURE 1A&B). Flow cytometry confirmed a graded expansion of ITGB8+ (cluster 0) lining fibroblasts and a contraction of DPP4+ (cluster 1) sub-lining fibroblasts with OA progression (FIGURE 1D&E). A pathway analysis was performed on human fibroblast subclusters highlighting that cluster 0 and 1 are primarily involved in pathways related to the extracellular matrix (ECM)(FIGURE 1C). To confirm these findings, we conducted bulk RNA sequencing on KL1 and KL3/4 graded human synovium, confirming that genes upregulated in late-stage synovial samples show enrichment for matrisome-related genes (FIGURE 1F&G). A regulatory transcription factor (TF) prediction analysis was performed on ECM genes from the DEG lists of human clusters 0 and 1 using Catrin. This identified three putative upstream TFs, namely PGR, ELF1 and BHLHE40, specifically associated with late-stage cluster 0. To profile the temporal dynamics of key cell subtypes the DMM mouse model characterized the presence of cell subsets and their transcriptomic profiles analogous to those found in human synovia. The snRNA sequencing of 19304 nuclei from DMM/naïve mouse synovium revealed comparable fibroblast subtypes to those identified in humans. Specifically, the largest late-stage human fibroblast subcluster (cluster 0) was most similar to mouse cluster 1, while the prevalent early-stage human fibroblast subcluster (cluster 1) most resembled mouse fibroblast cluster 4 (F1GURE 1H-J). Conclusion: We have identified an endophenotypic shift in fibroblast subsets from early to late stages of radiographic knee OA that may play a crucial role in synovial ECM regulation and fibrosis during OA. Subsequent investigations will elucidate if the identified TF's control ECM regulation and fibrosis through in vitro and in vivo knock out models. REFERENCES: NIL. Acknowledgements: This work is funded by the Canadian Institute of Health Research Operating Grant, Tony and Shari Fell Platinum Chair in Arthritis Research and Canada Research Chairs Program. Disclosure of Interests: None declared.

Abstract Metabolic mediators play an important role in regulating inflammation(1). Rheumatoid arthritis and spondyloarthritis are common inflammatory diseases of the joint, aggravated in context of obesity(2). These patients experience systemic bone loss(3,4), which is not sufficiently controlled by disease-modifying therapeutics, despite adequate control of inflammation(5,6). Here we report an unexpected role for GDF15 (Growth Differentiation Factor 15), a central mediator of food intake(7–10), in inflammation-associated bone loss. Serum GDF15 levels were found to be elevated in arthritis patients and inversely correlated with bone density. However, GDF15 itself does not appear to promote arthritis. Rather, GDF15 mediates trabecular bone loss through its receptor GFRAL, which is expressed exclusively in the hindbrain(7–10). GDF15-GFRAL binding results in β-adrenergic activation of bone Marrow Adipogenic Lineage Precursors (MALPs), mesenchymal cells which are known to stimulate osteoclasts and trigger bone loss(11–14). These data demonstrate how a metabolic mediator controls bone loss through a brain-bone axis in inflammatory diseases. These findings may lead to more specific therapeutic interventions to protect bone through targeting GDF15 or MALPs.

OBJECTIVES:In this study, we employ a multiomic approach to identify major cell types and subsets, and their transcriptomic profiles within the infrapatellar fat pad (IFP), and to determine differences in the IFP based on knee osteoarthritis (KOA), sex and obesity status. METHODS:Single-nucleus RNA sequencing of 82 924 nuclei from 21 IFPs (n=6 healthy control and n=15 KOA donors), spatial transcriptomics and bioinformatic analyses were used to identify contributions of the IFP to KOA. We mapped cell subclusters from other white adipose tissues using publicly available literature. The diversity of fibroblasts within the IFP was investigated by bioinformatic analyses, comparing by KOA, sex and obesity status. Metabolomics was used to further explore differences in fibroblasts by obesity status. RESULTS:We identified multiple subclusters of fibroblasts, macrophages, adipocytes and endothelial cells with unique transcriptomic profiles. Using spatial transcriptomics, we resolved distributions of cell types and their transcriptomic profiles and computationally identified putative cell-cell communication networks. Furthermore, we identified transcriptomic differences in fibroblasts from KOA versus healthy control donor IFPs, female versus male KOA-IFPs and obese versus normal body mass index (BMI) KOA-IFPs. Finally, using metabolomics, we defined differences in metabolite levels in supernatants of naïve, profibrotic stimuli-treated and proinflammatory stimuli-treated fibroblasts from obese compared to normal BMI KOA-IFPs. CONCLUSIONS:Overall, by employing a multiomic approach, this study provides the first comprehensive map of the cellular and transcriptomic diversity of human IFP and identifies IFP fibroblasts as key cells contributing to transcriptomic and metabolic differences related to KOA disease, sex or obesity.

This is the first comprehensive study of the impact of biodegradation on the structure, surface potential, mechanical and piezoelectric properties of poly(3‐hydroxybutyrate) (PHB) scaffolds supplemented with reduced graphene oxide (rGO) as well as cell behavior under static and dynamic mechanical conditions. There is no effect of the rGO addition up to 1.0 wt% on the rate of enzymatic biodegradation of PHB scaffolds for 30 d. The biodegradation of scaffolds leads to the depolymerization of the amorphous phase, resulting in an increase in the degree of crystallinity. Because of more regular dipole order in the crystalline phase, surface potential of all fibers increases after the biodegradation, with a maximum (361 ± 5 mV) after the addition of 1 wt% rGO into PHB as compared to pristine PHB fibers. By contrast, PHB‐0.7rGO fibers manifest the strongest effective vertical (0.59 ± 0.03 pm V −1 ) and lateral (1.06 ± 0.02 pm V −1 ) piezoresponse owing to a greater presence of electroactive β ‐phase. In vitro assays involving primary human fibroblasts reveal equal biocompatibility and faster cell proliferation on PHB‐0.7rGO scaffolds compared to pure PHB and nonpiezoelectric polycaprolactone scaffolds. Thus, the developed biodegradable PHB‐rGO scaffolds with enhanced piezoresponse are promising for tissue‐engineering applications.

SUMMARY Metabolic mediators play an important role in regulating chronic inflammation in the body. Here we report an unexpected role for GDF15 (Growth Differentiation Factor 15), a central mediator of food intake, in inflammation-associated bone loss. GDF15 serum levels were found to be elevated in arthritis patients and inversely correlated with bone density. Despite being associated with inflammation, we found that GDF15 itself does not cause, nor contribute to, clinical or histopathological arthritis. Rather, under inflammatory conditions, GDF15 mediates trabecular bone loss through its receptor GFRAL, which is exclusively expressed in the hindbrain. GDF15-GFRAL binding results in β-adrenergic activation of MALPs (Marrow Adipocytic Lineage Precursors) in the bone marrow, which stimulate osteoclasts and trigger bone loss. These data suggest a metabolic mediator-controlled brain-bone axis in inflammation, through which bone loss is induced in a contextual rather than general manner. These findings may lead to more specific therapeutic interventions to protect bone.

Objective Divergent therapeutic outcomes on different disease domains have been noted with IL-23 and IL-17A-blockade in PsA. Therefore, elucidating the role of ROR gamma t, the master regulator of type 17 immune responses, is of potential therapeutic interest. To this end, ROR gamma t inhibition was assessed in combined skin, joint and gut inflammation in vivo, using a PsA model. Methods We tested the efficacy of a ROR gamma t antagonist in B10.RIII mice challenged with systemic overexpression of IL-23 by hydrodynamic injection of IL-23 enhanced episomal vector (IL-23 EEV). Clinical outcomes were evaluated by histopathology. Bone density and surface erosions were examined using micro-computed tomography. Cytokine production was measured in serum and by intracellular flow cytometry. Gene expression in PsA-related tissues was analysed by qPCR. Results ROR gamma t-blockade significantly ameliorated psoriasis, peripheral arthritis and colitis development in IL-23 EEV mice (improvement of clinical scores and weight loss respectively by 91.8%, 58.2% and 7.0%, P < 0.001), in line with profound suppression of an enhanced type IL-17 immune signature in PsA-affected tissues. Moreover, inflammation-induced bone loss and bone erosions were reduced (P < 0.05 in calcaneus, P < 0.01 in tibia). Sustained IL-23 overexpression resulted in only mild signs of sacroiliitis. Gamma-delta (gamma delta)-T cells, the dominant source of T cell-derived IL-17A and IL-22, were expanded during IL-23 overexpression, and together with Th17 cells, clearly countered by ROR gamma t inhibition (P < 0.001). Conclusion ROR gamma t-blockade shows therapeutic efficacy in a preclinical PsA model with protection towards extra-musculoskeletal manifestations, reflected by a clear attenuation of type 17 cytokine responses by gamma delta-T cells and Th17 cells.

Background Osteoarthritis (OA) is a complex, multifactorial and heterogeneous joint disease of unknown etiology. OA research has largely focused on articular cartilage degeneration with little attention given to other joint tissues, including the synovium. The synovium lines the joint capsule, produces synovial fluid for lubrication, and is emerging as a contributor to OA pathogenesis. In OA, the synovium exhibits increased vascularization, inflammation, hyperplasia and fibrosis. Synovial cells that contribute to these pathological events during early and advanced stages of OA are not well characterized. The emergence of RNA sequencing (RNAseq) at the resolution of a single cell or nucleus allows for the identification of distinct cells that may contribute to OA pathogenesis. Objectives To delineate the synovium’s role in OA pathogenesis we sought to identify if distinct cell subtypes exist in the synovium of early (KL1) versus late stages (KL3/4) of radiographic knee OA using single nucleus RNA sequencing. Methods Synovia from patients with early (KL=I; n=5) and late (KL =III/IV; n=4) stage radiographic knee OA were subjected to single nucleus (sn)RNAseq and to bioinformatics analyses. Canonical cell-specific markers were used to identify cell types from the unsupervised clustering analysis and prominent cell types were re-clustered. Differentially expressed gene (DEG) lists between the subclusters were determined based on top gene expression within a cell type between early and late OA synovium. Cell surface markers identified from the DEGs were validated by immunohistochemistry. Pathway and gene ontology enrichment analysis were performed on fibroblast subclusters to identify prominent pathways and transcription factors that were upstream regulators. Ongoing in vivo and in vitro methods are being used to assess these transcription factors in both fibroblast cell culture and an OA mouse model. Results Fibroblasts and macrophages constituted 75% of the cells from early and late-stage synovium and re-clustering analysis resolved 8 fibroblast and 6 transcriptionally distinct macrophage subclusters (Figure 1). Cluster based nuclei proportion differences identified fibroblast clusters 1, 2, 4 and 6 and macrophage clusters 1, 2 and 5 to contribute to early-stage samples while fibroblast clusters 0, 3 and 5 and macrophage clusters 0, 3 and 4 to late-stages. Downstream analyses focused on fibroblasts and putative cell surface markers from fibroblast subclusters were identified from DEGs and confirmed by immunohistochemistry. The fibroblast subclusters were subjected to pathway analyses which identified clusters 0 and 1 to be the most prominent clusters which both shared common ECM related pathways. Upstream transcription factors that regulate ECM related genes were identified for both subclusters 0 and 1. Current efforts are focussed on selecting and targeting transcription factor(s) for both in vitro and in vivo analyses by utilizing siRNA’s in fibroblast culture and a cre-lox mouse system to identify the mechanisms associated with the synovial pathology during OA. Conclusion SnRNAseq analysis identified distinct subclusters of fibroblasts and macrophages to exist in human OA knee synovia. Certain subclusters were more representative of the early stage while others were more representative of the late stage of the disease. Further validation studies are being performed to assess the functional roles of these subclusters and whether targeting them would attenuate disease progression. KT and EG share equal first author contribution. DE and Mk share equal senior author contribution. Acknowledgements Canadian Institute of Health Research. Disclosure of Interests None Declared.

Arthritis is the most common extra-intestinal complication in inflammatory bowel disease (IBD). Conversely, arthritis patients are at risk for developing IBD and often display subclinical gut inflammation. These observations suggest a shared disease etiology, commonly termed "the gut-joint-axis." The clinical association between gut and joint inflammation is further supported by the success of common therapeutic strategies and microbiota dysbiosis in both conditions. Most data, however, support a correlative relationship between gut and joint inflammation, while causative evidence is lacking. Using two independent transgenic mouse arthritis models, either TNF- or IL-1β dependent, we demonstrate that arthritis develops independently of the microbiota and intestinal inflammation, since both lines develop full-blown articular inflammation under germ-free conditions. In contrast, TNF-driven gut inflammation is fully rescued in germ-free conditions, indicating that the microbiota is driving TNF-induced gut inflammation. Together, our study demonstrates that although common inflammatory pathways may drive both gut and joint inflammation, the molecular triggers initiating such pathways are distinct in these tissues.