Macrophages are critical drivers of synovial inflammation in Rheumatoid Arthritis

Macrophages are an exquisitely plastic pool of innate cells critically involved in directing the immune response in RA. Upon entry into the synovium, peripheral blood monocytes differentiate into the dichotomous M1 (pro-inflammatory) or M2 (anti-inflammatory) macrophage activation states. However, it is now known that macrophages are heterogeneous both in function and origin with many macrophages seeded during embryonic development independently. Despite this, the precise nature and function of infiltrating monocyte-derived macrophages and their precursor cells are poorly defined in RA with even less known about RA synovial tissue macrophages. Therefore, the aims of this thesis are to elucidate the distinct pathogenic functions, bioenergetic demands and activation status of monocytes, monocyte-derived macrophages and synovial tissue macrophage subsets in RA. In this study we demonstrate that circulating RA CD14+ monocytes are primed to produce pro-inflammatory mediators, a phenotype indicative of M1-like macrophage polarisation. Metabolic analysis of RA monocytes using the Seahorse Flux analyser reveals a robust boost in both OXPHOS and glycolysis in RA CD14+ monocytes compared to HC. Interestingly, the hyper-inflammatory, hyper-metabolic phenotype of RA monocytes persists following differentiation into ex vivo macrophages. RA M1 macrophages replicate the inflammatory memory bias of their precursor cells demonstrating heightened glycolysis and mitochondrial respiration coupled with altered mitochondrial morphology compared to HC. In addition, we demonstrate a consistent upregulation of glycolytic machinery indicating fundamental abnormalities in glucose processing in RA myeloid cells. Furthermore, analysis of polarised RA M1/M2 macrophages reveals divergent inflammatory, bioenergetic and phagocytic functions. Marked transcriptional variance was indicated by RNA-seq, with a key role for STAT3 activation in macrophage polarisation identified. Mechanistically, we demonstrate that the hyper-inflammatory and metabolic phenotype of RA monocytes and M1 macrophages, is mediated through STAT3 phosphorylation whereby inhibition of STAT3 activity switches the pathogenic phenotype of myeloid cells for resolution of inflammation. Distinct subsets of tissue macrophage in the inflamed RA synovium remain largely unexplored. In this thesis we phenotypically characterise RA synovial tissue macrophages revealing a spectrum of macrophage activation states that don?t conform to the binary M1/M2 framework in vivo. Within this spectrum we identify for the first time, that the dominant macrophage population residing in the RA synovium is a transitional subset of tissue-resident CD206+CD163+ macrophages that display elevated CD40 expression. We demonstrate that this subset is enriched in synovial tissue compared to fluid but specifically in RA synovium compared to OA and PsA synovial pathotypes. Furthermore, the CD206+CD163+ macrophage subset is present in healthy synovial tissue but does not express CD40. CD206+CD163+ and CD206-CD163- macrophage populations were sorted from RA synovial tissue and synovial fluid and RNA-seq analysis performed. We reveal that the CD206+CD163+ macrophage population is transcriptionally distinct from synovial fluid, double negative CD206-CD163- and pure monocyte-derived M1/M2 macrophages, with unique tissue-resident gene signatures. Moreover, we demonstrate differing metabolic demands between CD206+CD163+ and CD206-CD163- macrophage subsets using FLIM analysis. Finally, CD206+CD163+ macrophages induce enhanced autologous T cell responses and spontaneously secrete high levels of IL-8, IL-6 and TNFa. Finally, the effect of key macrophage-secreted cytokines, OSM and TNFa on stromal cell (HUVEC/RAFLS) function was examined. OSM differentially regulates pro-inflammatory mechanisms, significantly promotes pro-angiogenic and invasive mechanisms and alters cellular bioenergetics in favour of glycolysis in RAFLS and HUVEC. Moreover, while OSM synergises with TNFa to differentially regulate pro-inflammatory mechanisms in both cell types, synergistic regulation of metabolic reprogramming is observed only in RAFLS, and not HUVEC. Finally, we demonstrate that OSM in combination with TNFa induces transcriptional activity of STAT3 in RAFLS specifically. Taken together, this data provides a greater understanding of the critical role both infiltrating and tissue-resident macrophages play in perpetuating inflammation in RA. Further investigation of the molecular patterns and cues that shape specific synovial macrophage subsets may provide opportunities to reinstate RA joint homeostasis