Defective Macrophage Plasticity Exacerbates Progression of Inflammation in Patients with Rheumatoid Arthritis

Background Rheumatoid Arthritis (RA), along with other inflammatory arthritides, affects over 1% of the global population. There are no cures, and only 20% of patients achieve 2-year drug-free remission. Importantly, recent studies show that macrophages have a dual role in synovial inflammation due to the existence of proinflammatory subsets (M1) and remission-associated macrophages (M2), with several M2 subtypes reported in the literature (M2a/M2b/M2c/M2d). Aberrant macrophage plasticity programming may contribute to RA pathogenesis through the secretion of proinflammatory cytokines and chemokines, promoting joint and tissue degradation, bone erosion, and disease progression. Methods Peripheral blood CD14 + cells were isolated from patients with RA and healthy controls and polarised towards M1, M2a, M2b, M2c and M2d macrophage subsets under atmospheric oxygen and hypoxic conditions simulating the inflamed joint. PCR and multiparametric flow cytometric analysis were used to examine expression of macrophage subset-specific markers (CD14, CD11c, CD38, CD86, CD163, CD206, CD209, MerTK, IRF4) as well as chemokine receptors CCR1, CCR2, CCR4, CCR5 and CCR8. Flow cytometric-based protein synthesis analysis was used to assess metabolic pathways. scRNAseq of synovial tissue macrophages from patients with RA and healthy controls validated the primary data. Macrophage subtypes were also examined for in-vitro differentiation into chondrocytes. Results scRNA sequencing analysis (GSE200815) identified M1, M2a, M2b, M2c and M2d clusters with surprisingly comparable frequency between HCs and patients with RA; however, global and subtype-specific differences promote inflammation, with increased CD86, VEGF and reduced MerTK expression by RA M2 subsets. In vitro polarised RA macrophages displayed altered metabolism and increased CD86, CD38 and reduced MerTK expression especially from M2c macrophages compared to HCs. Hypoxic conditions exacerbated the differences between HC and RA M2c macrophages, potentially driving the inflammatory process in the arthritic joint. Except for M2c macrophages all other subsets could be differentiated to osteoclasts with RA macrophages derived osteoclasts leading to significantly more and potentially pathogenic cells. Conclusion Dysregulated phenotypic, metabolic and functional plasticity displays intermediate phenotypes in macrophages, which supports disease progression to chronic inflammation in patients with RA.