POS0443 INSIDE THE JOINT OF INFLAMMATORY ARTHRITIS PATIENTS: HANDLING AND PROCESSING OF SYNOVIAL TISSUE BIOPSIES FOR HIGH THROUGHPUT ANALYSIS

Abstract

BackgroundInflammatory arthritis (IA) is a chronic systemic autoimmune disease of unknown aetiology, which affects the joints. While studies of immune cell populations in peripheral blood have been informative regarding potential immune cell dysfunction and possible patient stratification, there are considerable limitations in identifying the early events that lead to synovial inflammation. The joint, as the site of inflammation and the local microenvironment, exhibit unique characteristics that contribute to disease pathogenesis. The limited availability of synovial tissue (ST) biopsies is a key incentive for the utilisation of high-throughput techniques in order to maximise information gain.ObjectivesThis work aims to provide an overview of key methods and novel techniques that are used in the handling, processing and analysis of ST biopsies and the potential synergy between these techniques.MethodsWe describe the utilisation of high dimensionality flow cytometric analysis, single cell RNA sequencing, ex vivo functional assays, including T cell activation, endocytosis and two-photon fluorescent lifetime imaging microscopy (FLIM).ResultsWhen comparing different methods for ST cell suspension generation we observed that the combination of mechanical and enzymatic digestion resulted in the release of considerably higher numbers of total viable cells when compared to mechanical digestion alone, although consideration should be taken in the cleaving of extracellular markers, like CD27. We next compared two different cryopreservation methods to that of freshly digested ST and observed similar viability and frequency of immune cells. Functional characterisation of ST cells can be challenging due to the high number of cells required for analysis, herein, we utilised the above protocols to establish ST viable cell suspensions and optimised different experimental approaches for phenotypical/functional characterisation. To investigate the functional consequence of OxPhos inhibition on ST T-cell polyfunctionality, ex-vivo ST cell suspensions from IA patients were stimulated with PMA/Ionomycin in the presence/absence of FCPP followed by metabolic profile characterisation via FLIM and high dimensionality flow-cytometric analysis for T cell-derived cytokines. Treatment with FCPP resulted in a decreased in T-cell polyfunctionality specifically in co-expression of TNF-α,-IL-2,-IFN-γ,IL-17A, -GM-CSF, an effect associated with a shift in their metabolic profile. In addition, freshly digested ST cell suspensions were subjected to an optimized assay to evaluate endocytosis in multiple populations simultaneously without the need for cell sorting. Briefly, digested cells were incubated in parallel at 4°C (passive endocytosis) and 37°C (active endocytosis) with DQ OVA(Ovalbumin). ST cells were then stained for multiple populations, demonstrating differential endocytosis capacity across pathotypes and disease controls.Finally, utilisation of novel bioinformatics analysis of RNAseq a data showed differential gene expression and pathway enrichment involved in IA pathogenesis and allowed for the comparison of cell specific enrichment scores and transcription factor usage based on pathotype and gender.ConclusionThe introduction of new powerful techniques in the study of ST inflammation, brings new challenges and significant opportunities. These approaches will accelerate our path towards understanding of the mechanisms involved in the pathogenesis of IA and lead to the identification of new avenues of therapeutic intervention.AcknowledgementsWe would like to thank all the patients who consented to be involved in this study.Disclosure of InterestsNone declared.