Pos0056 global steroid metabolism in macrophages: shaping inflammatory function and disease activity in rheumatoid arthritis

BackgroundMacrophages are key drivers of joint destruction and disease pathophysiology in rheumatoid arthritis (RA), where their inflammatory function is influenced by steroid hormones such as androgens and glucocorticoids (GCs). Local bioavailability of these steroids is determined by both systemic adrenal/gonadal synthesis and local metabolism in peripheral target tissues. The inflammatory regulation and function of steroid hormone metabolism by key rate limiting enzymes in chronic inflammatory diseases such as RA remain poorly defined and could present new therapeutic targets.ObjectivesCharacterise regulation of global steroid metabolism in macrophages in RA and determine its contribution to androgen and GC availability, macrophage function and disease activity.MethodsBulk and single cell RNA-sequencing of FACS-sorted macrophages were analysed using previously published datasets from RA patients (27 female, 8 male)(1, 2). Gene expression of rate limiting steroid metabolism enzymes were assessed in macrophages and their subsets and correlated to clinical parameters of disease activity. Primary human monocyte-derived macrophages were polarised to non-inflammatory (M-CSF 20ng/ml) and inflammatory activated (M-CSF 20ng/ml, IFNγ 20ng/ml, TNFα 10ng/ml) subsets and treated with active or inactive metabolites of GCs (cortisol/cortisone 100nmol/l) and androgens (androstenedione/testosterone/DHEA 100nmol/l; DHT 10nmol/l). Metabolism and functional effects were assessed in primary cultures and RA synovial fluids by liquid chromatography mass spectrometry, RT-qPCR and ELISA.ResultsSignificant differentially expressed genes (DEGs) were identified in the GC and androgen metabolism pathways in synovial macrophages when stratified for high and low disease activity by DAS28-CRP. Expression of the GC-activating enzyme HSD11B1 and androgen activating enzyme SRD5A1 were significantly increased and positively correlated with disease severity. The androgen activating enzyme AKR1C3 was significantly suppressed and negatively correlated with disease severity. SRD5A1 and HSD11B1 expression were localised to S100A12pos and SPP1pos subsets associated with active RA, whilst AKR1C3 was primarily expressed by MerTKposTREM2high subsets associated with RA remission. Inflammatory activation of primary macrophages decreased AKR1C3, and increased HSD11B1 and SRD5A1 expression. This resulted in a shift in intracrine production of active GCs and androgens favouring increased levels of the active GC cortisol and the potent androgen DHT. The resulting changes in steroid ratios in inflammatory activated macrophages resulted in lower expression and release of the pro-inflammatory mediators TNFα, IL6 and IL12 indicating functional significance. In vivo, metabolic changes favouring increased GC activation and reduced androgen activation correlated with disease severity determined by DAS28-CRP.ConclusionWe have shown for the first time a role for macrophages and their tissue subsets in the inflammatory metabolism and activation of GCs and androgens in RA, which influence macrophage function and disease activity. Targeting these key metabolic pathways represents a novel route to modifying and suppressing disease activity and joint destruction in chronic polyarthritis.References[1]Zhang F, Wei K, Slowikowski K, Fonseka CY, Rao DA, Kelly S, et al. Defining inflammatory cell states in rheumatoid arthritis joint synovial tissues by integrating single-cell transcriptomics and mass cytometry. Nature Immunology. 2019;20(7):928-+.[2]Alivernini S, MacDonald L, Elmesmari A, Finlay S, Tolusso B, Gigante MR, et al. Distinct synovial tissue macrophage subsets regulate inflammation and remission in rheumatoid arthritis. Nature Medicine. 2020;26(8):1295-+.AcknowledgementsThis research was funded by the Wellcome Trust (ref: 215243/Z/19/Z)Disclosure of InterestsNone declared