TBA (17A186)

Distinct macrophage phenotype and bioenergetic profiles in Rheumatoid Arthritis

Author(s)

Megan Hanlon1, Trudy McGarry2, Mary Canavan1, Candice Lowe2, Siobhan Wade1, Douglas J. Veale2, Ursula Fearon1. 

Department(s)/Institutions

1Molecular Rheumatology, Trinity Biomedical Sciences Institute, Trinity College Dublin. 2Centre for Arthritis and Rheumatic Diseases, St. Vincent's University Hospital, Dublin. 

Introduction

Diversity and plasticity of macrophage subsets within the joint has yet to be explored. The concept of macrophage polarization into M1 inflammatory macrophages (or classically activated) and M2 tissue resolving macrophages (or alternatively activated) parelled by changes in the bioenergetic cell profile has received much attention in recent years. 

Aims/Background

We therefore aimed to examine the phenotype and metabolic profile of M1 and M2 macrophages in the inflamed RA joint. 

Method

Blood was obtained from healthy and RA donors, PBMC isolated CD14+ cells sorted and differentiated into macrophages in the presence of M-CSF for 8 days. Macrophages were polarised to either M1 (LPS and IFNγ) or M2 (IL-4). Markers of in vitro polarisation KLF6, PPARG, TGM2 and STAB1 and of metabolism HIF1α, HK2, LDHA, PFKFB3, G6PD, PDK1 and PDK2 were quantified by Real Time-PCR. Seahorse XFE technology was utilised to measure the two major energy-using pathways, glycolysis (ECAR) and oxidative phosphorylation (OCR). Finally, to phenotype macrophages in the RA joint, synovial tissue was digested to yield a single cell suspension which was subsequently stained with a panel of fluorochrome antibodies (CD68, CD45, CD40, CD253, CD163, CD206), acquired by multicolour flow cytometry and analysed using FlowJo software. preprocess

Results

M1 macrophages were confirmed by increased expression of KLF6 and PPARG while M2 macrophages expressed high TGM2, PPARG and STAB1. M1 cells had significantly higher expression of pro-glycolytic genes HIF1α, HK2, LDHA and PFKFB3, all of which were deficient in M2 macrophages (All p<0.05) and significantly higher than their healthy counterpart, suggesting a greater glycolytic profile in RA-derived cells G6PD was significantly decreased in M1 and increased in M2 macrophages, with PDK enzymes 1/2 decreased in M1 (both p<0.05). Seahorse technology demonstrated that M1 macrophages have higher baseline ECAR and lower OCR, whereas M2 macrophages tend to utilise oxidative phosphorylation more readily. This was paralleled by higher pro-inflammatory cytokines levels (IL-6, IL-8, OSM) in M1 vs M2 macrophages. Finally, RA ST analysis determined that approximately 40% of infiltrating CD45+ immune cells are positive for the pan-macrophage marker CD68. Interestingly, the classical paradigm of M1 and M2 macrophages is not found in the RA synovium. Instead, a spectrum of macrophages which express both M1 and M2 markers were identified. Specifically, 35-55% of ST macrophages express M2 markers CD206 and CD163. However these cells also have high expression of M1 markers CD40 and 253, indicating that the unique inflammatory environment of the RA synovium maintains M1 like macrophages with M2 like properties. preprocess

Conclusions

This study demonstrated distinct metabolic profiles in M1 and M2 RA macrophages, demonstrating their opposing roles in perpetuating and resolving inflammation, respectively. Furthermore, we have identified for the first time, a spectrum of tissue-specific macrophages which have both an M1-like and M2-like phenotype, suggesting that RA joint macrophages remain plastic and function according to their surrounding microenvironment. preprocess