Title: Dissecting the roles of synovial tissue macrophage subpopulation(s) in reinstating joint immune-homeostasis in Rheumatoid Arthritis
Funder: Versus Arthritis

Abstract: Background: Rheumatoid Arthritis (RA) therapies target mainly inflammatory mediators delivering significant benefit, however, partial or non-response remains a significant challenge. Drug-free remission is uncommon but does occur; providing proof-of-concept that articular immune-homeostasis can be reinstated. Our single-cell phenotypic and transcriptomic analyses of synovial tissue macrophages (STMs) from biopsies of RA patient in remission identified a novel MerTK/TREM2/TIM4pos STM subpopulation. These occupy a distinct developmental-trajectory branch compared to monocyte-derived inflammatory STMs and have a transcriptomic phenotype suggesting they can mediate synovial inflammatory resolution. Our hypothesis is that these cells can be specifically directed, in a therapeutic context, to resolve inflammation and reinstate homeostasis. Aim and importance: We will characterise STM-driven cellular and molecular mechanisms that mediate or maintain remission in RA. Deeper understanding of molecular pathways that determine STM functions will generate therapeutic strategies to redirect their functions towards endogenous resolution of inflammation, reinstating synovial homeostasis, and the prospect of cure. Experimental plan: We will examine (i) how STM subpopulations regulate autoimmune and danger-signal induced cellular activation within the synovium, (ii) the reciprocal cellular cross-talk between STM subpopulations and synovial fibroblasts that informs their phenotype and effector functions. To uncover the functional profile of the newly-identified TREM2/TIM4posSTM subpopulation, we will compare their regulatory properties with other STM subpopulations by evaluating their responses to ACPA-complexes and DAMPs, and their impact on the activation of synovial fibroblasts. To achieve that we will use multiplex assays for soluble mediators and multi-omics single-cell profiling of ex-vivo STM cultures, and co-cultures with fibroblasts. Emerging functional profiles will be then mapped in-situ using synovial tissue spatial transcriptomics. To identify molecular mechanisms that underlie regulatory functions of STMs, we will compare deep-transcriptomic profiles of TREM2/TIM4posSTMs with other STM subpopulations, and rank differentially expressed pathways by bio-informatic modelling, followed by functional validation of prioritised pathways by gene-gain and loss-of-function experiments in primary cells, and therapeutic testing in animal models. To study the differentiation stages of STMs towards regulatory phenotypes, we plan to establish stem-cell derived human STMs from embryonic macrophage precursors. Firstly, we will generate embryonic macrophage precursors (iMacs) from inducible pluripotent stem-cells (iPSC). We will then test the potential of iMacs to differentiate to STM-like phenotype (called inducible STMs; iSTMs) by providing the “tissue-specific cues” via co-culture with remission synovial fibroblasts. Emerging potential iSTM clusters will be identified by scRNAseq and compared with our scRNA-Seq data sets of primary STMs. Finally, we will determine the suitability of iSTMs for therapeutic modelling such as CRISPR-Cas9 gene-editing. This experimental plan will characterise the regulatory macrophage functions in the RA synovium and establish their potential for therapeutic targeting. Relevance to Versus Arthritis UK priorities and patient benefit: This study will address the therapeutic imperative by seeking pathways that establish immunological reset and therapeutic homeostasis with the prospect of cure. Revealing molecular pathways that subserve regulatory functions of STMs will identify therapeutic targets for future exploitation to resolve inflammatory arthritis. Such targets will be particularly attractive because they represent reinstating endogenous feedback regulation.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFLAMMATORY AND IMMUNE SYSTEM