SAT0007 Distinct macrophage phenotype and bioenergetic profiles in rheumatoid arthritis
Authors: M.M. Hanlon, T. McGarry, M. Canavan, C. Lowe, S. Wade, D.J. Veale, U. Fearon
Journal: Annals of the Rheumatic Diseases (2018)

<h3>Background</h3> Diversity of macrophage subsets within the joint remains unknown. The concept of macrophage polarisation into M1 inflammatory macrophages and M2 tissue-resolving macrophages, parelled by changes in the bioenergetic cell profile, has received much attention. Hence, we aimed to examine the phenotype of macrophages within the inflamed RA joint, along with the metabolic and inflammatory capacity of RA monocyte-derived macrophages compared to healthy individuals. <h3>Objectives</h3> To examine the phenotype, metabolic and inflammatory profile of pro-inflammatory and anti-inflammatory macrophages within the inflamed RA joint. <h3>Methods</h3> Blood obtained from healthy and RA donors, CD14 +cells sorted and differentiated into macrophages for 8 days. Macrophages were polarised to either M1 (LPS and IFNγ) or M2 (IL-4). Markers of polarisation, metabolism and inflammation were quantified by Real Time-PCR. Seahorse technology measured the major energy-using pathway, oxidative phosphorylation (OCR). Finally, synovial tissue (ST) was digested to yield a single cell suspension, this was then stained using a panel of fluorochrome antibodies (CD45, CD40, CD68, CD64, CD163, CD206, CD253), and subsequently analysed using FlowJo software. <h3>Results</h3> M1 macrophages were confirmed by increased expression of KLF6 while M2 macrophages expressed high TGM2, PPARG and STAB1). M1 cells had significantly higher expression of pro-glycolytic genes (HIF1α, HK2, LDHA, PFKFB3 and PKM2), which were deficient in M2 macrophages and higher compared to healthy control. This was paralleled by higher pro-inflammatory cytokines levels (IL-8, OSM, MCP-1, RANTES IRAK-1, CCR5 and SOCS3) in M1 vs M2 macrophages, with RA derived macrophages showing higher expressions of pro-inflammatory mediators compared to healthy control. G6PD, PHD3 and PDK1/2 were significantly decreased in M1 yet increased in M2 macrophages, and along with this seahorse technology demonstrated that M2 macrophages have higher baseline OCR. Finally, ST analysis determined approximately 40% of CD45 +cells are positive for the pan-macrophage marker CD68. Interestingly, the classical paradigm of M1 and M2 macrophages is not found. Instead, a spectrum of macrophages were identified with approximately 57% of ST macrophages expressing M2 markers (CD206, CD163) with the M1 activation marker CD40. <h3>Conclusions</h3> This study demonstrated distinct metabolic profiles in M1/M2 RA macrophages; their opposing roles in perpetuating and resolving inflammation, respectively. We have identified, for the first time, a transitionary subtype of tissuespecific macrophages, suggesting that these cells remain plastic and function according to their microenvironment This study demonstrated distinct metabolic profiles in M1/M2 RA macrophages; their opposing roles in perpetuating and resolving inflammation, respectively. We have identified, for the first time, a dominant transitionary subtype of tissue specific macrophages with at least six other phenotypically distinct subtypes also present, suggesting that these cells remain plastic and function according to their microenvironment. <h3>Disclosure of Interest</h3> None declared