TBA (18A168)

Distinct macrophage phenotype and bioenergetic profiles in Rheumatoid Arthritis

Author(s)

Megan M. Hanlon, Mary Canavan, Trudy McGarry, Candice Low, Douglas J. Veale, Ursula Fearon.

Department(s)/Institutions

Molecular Rheumatology, Trinity Biomedical Sciences Institute, Trinity College Dublin

Introduction

Synovial macrophages play a key role in RA disease progression, however, the diversity and plasticity of macrophage subsets and their metabolic profile within the joint has yet to be elucidated.

Aims/Background

To phenotype distinct macrophage subsets within the RA joint, and determine the metabolic, inflammatory and phagocytosis function of RA macrophages compared to healthy controls (HC).

Method

Synovial-tissue biopsies from RA, PsA and OA, obtained through arthroscopy, were digested to yield a single cell suspension. Biopsy suspensions and synovial fluid mononuclear cells were analysed using advanced flow-cytometry with the following antibody panel (CD40,-CD45,-CD64,-CD68,-CD163,-CD206,-CD253). CD14+ monocytes were sorted from RA and HC bloods and differentiated/polarized into M1/M2 macrophages. Inflammatory (IL-8,-MCP-1,-IL-1b,-CCR5,-IRAK1,-OSM) and metabolic (HIF1a,-PFKFB3,-PKM2,-LDHA,-HK2) markers were measured by RT-PCR, and phagocytosis by OVA luciferase-yellow assays. Glycolysis (ECAR) and oxidative phosphorylation (OCR) were quantified by Seahorse -XFE- technology.

Results

RA synovial-tissue and fluid CD68+ macrophages displayed markers typical of both M1(CD40+CD253+) and M2(CD206+CD163+). A significant increase in frequency of CD68+ and CD64+ macrophages in synovial-tissue compared to fluid was observed (p<0.05), with significant increases in marker expression of CD40,CD163,CD206 (p<0.07). A spectrum of macrophage-subtypes within the inflamed joint was observed, with significant enrichment of a dominant double positive CD206+CD163+ macrophage-subtype in the synovial-tissue versus synovial fluid demonstrated (p<0.05). Increased frequency of CD206+CD163+ macrophages and higher expression of activation marker CD40 were demonstrated in RA synovial-tissue compared to PsA and OA. M1 macrophages demonstrate a pro-glycolytic phenotype with significant increases in HIF1a,-HK2,-PKM2,-and PFKB3, compared to M2, effects exacerbated in RA macrophages compared to HC. In parallel, using seahorse-technology RA M1 and M2 macrophages displayed higher ECAR and OCR profiles, in addition to an increased ECAR:OCR ratio compared to HC (p<0.05), evidence that RA macrophages switch to a glycolytic profile. This was paralleled by increased intracellular-cytokine expression of IL-1b,-IL-6 and TNFa and gene expression of IL-8,-IL-1b, OSM and MCP-1 (p<0.05). Finally, phagocytic ability of RA M1 was impaired compared to HC.

Conclusions

We have identified, for the first time, a dominant macrophages subtype enriched in RA synovial-tissue. Furthermore, RA M1/M2 have distinct metabolic profiles associated with differences in key inflammatory mediators and phagocytic function.