OP0094 SPATIAL POSITIONING AND MATRIX PROGRAMS OF SYNOVIAL FIBROBLASTS UNDERPIN IMMUNE CELL ORGANISATION AND SYNOVIAL TISSUE PATHOTYPE

Background Rheumatoid arthritis (RA) is highly heterogenous with distinct cellular and molecular tissue pathotypes that associate with disease outcome[1]. The synovial membrane is the primary target tissue and undergoes significant architectural re-modelling in response to inflammation. While fibroblasts are known to shape immune cell compartmentalisation in secondary lymphoid organs, by producing distinct sets of chemokines and remodelling extracellular matrix (ECM)[2], the mechanisms underlying immune cell localisation in the synovium are poorly understood. Objectives To define the functional zonation of the inflamed synovium at the single cell and regional tissue level using spatial transcriptomics and multiplex imaging. Methods We performed oligonucleotide probe-based spatially resolved transcriptomics on synovial tissue samples obtained by minimally invasive, ultrasound guided synovial tissue biopsies performed in patients with both rheumatoid arthritis (RA) and osteoarthritis (OA) using the 10x Visium platform. A total of 27 tissue biopsies processed sequencing 34170 spot transcriptomes. Each tissue section was composed on 1-8 fragments of synovial tissue from the same donor biopsy sample within each capture area, to maximise tissue representation and account for sampling variability. Cellular deconvolution of spot transcriptomes was performed using single cell reference datasets. Multiplex imaging was obtained to confirm gene profiles and cell types associated with the specific zonation patterns. Results We identified six spatially resolved tissue niches (or zones) within synovial membrane defined by gene expression and comprised of distinct populations of stromal and immune cells. Specific patterns of gene expression were observed in each of these sub-synovial tissue zones that link to functions of immune infiltrate recruitment and organisation. This zonation pattern is maintained across pathotypes (Fibroid, Lymphocytic) and spatial gene expression analysis revealed that RA is associated with specific synovial re-modelling and the formation of lymphocytic niches when compared to OA synovial tissue. We next defined zonation areas by manually annotating immune infiltrates (as aggregates) and vasculature, based on histology. From these, we selected fibroblasts specific to immune aggregate zones and peri-vascular zones (Figure 1). We have identified differences in gene expression between these fibroblasts based on their anatomical location. Peri-vascular fibroblasts have gene expression profiles related to myofibroblast differentiation, ECM deposition and modulation (COMP), but also immune cell recruitment (PLA2G2A), cell activation (FOS & FOSB), and lipid metabolism (APOD), also associated with OA synovium. These fibroblasts display either a TGF-β response or NOTCH3 activated[3] gene expression program and are expanded in the fibroid pauci-Immune) tissue pathotype. Immune interacting (peri-aggregate) fibroblasts in contrast, show specific gene expression profiles linked to ECM regulation (POSTN), degradation (MMP3) and co-ordination of ECM remodelling to recruit immune cells (SPARC), and permit aggregate formation. These fibroblasts display a T-cell interacting[4] and IFN-γ gene expression program and along with IL-1β and TNF-α activated lining layer fibroblasts dominate the stromal cell landscape of the lymphoid tissue pathotype. Conclusion We demonstrate that cytokine directed fibroblast-immune cell crosstalk, positional identity, and matrix specific, fibroblast gene expression programs are responsible for localising infiltrating immune cells to specific niches within the tissue and defining functional tissue zonation. These data identify novel therapeutically tractable disease pathways that underpin the pathogenicity of synovial tissue fibroblasts in RA. References [1]Lewis et al. Cell Reports 2019;28:2455-2470 [2]Bajénoff M et al. Immunity 2006;25:989–1001 [3]Wei K et al. Nature 2020;582:259-264. [4]Korsunsky I et al. Med (N Y) 2022;3:481-518.e14 Acknowledgements: NIL. Disclosure of Interests None Declared.