Does differential senescence between fibroblast subsets explain the increased progression towards Rheumatoid Arthritis and Osteoarthritis with age?

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This PhD studentship has three aims:Aim1: Molecular characterization of a panel of senescence markers in synovial fibroblast lining and sub-lining layer fibroblast subsets in sex matched patients across the spectrum of ages (20ys-80ys) with OA and RA Aim 2: Bioinformatic analysis of the relationship between the senescence hallmarks in lining and sub-lining fibroblasts in human fibroblast subsets compared to subsets analysed from established mouse models of inflammatory arthritis (CIA) and degenerative arthritis the destabilization of medial meniscus (DMM)Aim 3: Establish the anatomical localization of senescent fibroblast subsets in OA and RA fibroblast synovium compared to CIA and DMM synovium using multiplex immunofluorescence (CODEX) and RNA scope analysisObjective 1: We will digest human RA and OA synovium as described [3] over a range of ages (50-80) and following FACS sorting using CD45/THY1/PDPN to identify Lining Layer (LL), sub-lining (SL) and pericytes (PC) we will use test a panel of known senescence hallmarks for differential expression in these three subsets. These include mitochondrial health and ROS by FACS [4], lysosomal health (lipofuscin by SenTraGor or IHC, lysosomal function by 14C valine protein degradation assay (Wade-Martins, Oxford), DNA damage, protein damage by mass spec (Kessler, Oxford), SASP (senescence associated secretory phenotype, by intracellular FACS & ELISA) replicative senescence and autophagic flux (as described [5]). We know from data in the NIH AMP study, and previous tissue-based studies [6] that a minimum of 20 patient samples from OA and RA will provide sufficient power to demonstrate differences between groups of patients over three independent decades; 50-60yrs, 60-70yrs, 70-80yrs. Clinical data will be correlated with laboratory findings using tranSMART: An Open Source and Community-Driven Informatics and Data Sharing Platform for Clinical and Translational Research in which our clinical databases are housed. Objective 2: The functional relevance of findings Objective 1 will be assessed by comparison with current human single-cell datasets from the NIH Accelerating Medicine Partnership (AMP) to which we have contributed [6] with datasets generated from mouse models of RA (CIA) and OA (DMM) that are available in Oxford. In addition, explicit cross-species alignment of mouse and human fibroblasts will be performed in order for unbiased assessment of the existence of conserved fibroblast subtypes relating to senescence hallmarks. Objective 3: We have experience in the development and application of multi-parameter immunohistochemistry (using CODEX (up to 40 parameters) imaging system and confocal imaging Zeiss 880 Airyscan (up to 8 parameter, high spatial resolution) and RNAscope to develop spatial maps of cellular location with gene expression in the synovium. Utilizing this approach, we will verify the location of key senescence hallmarks identified in Objective 1 in tissue sections from human OA and RA synovial tissue as well as mouse CIA and DMM. We use a combination of FFPE and frozen sections to validate gene expression cassettes identified. Image analysis will be done using a combination of commercial (Imaris, CODEX Analysis Pipeline, Zen) and open source image analysis packages (ImageJ, CellProfiler).