ISR Autumn Meeting 2019

2nd Place Scientific Oral Presentation

Megan Hanlon

TBA (19A138)

Distinct monocyte and macrophage inflammatory and bioenergetic profiles in Rheumatoid Arthritis

Author(s)

Hanlon MM (1), McGarry T (1) , Canavan M(1) , Low C (2), Wade S (1), Nagpal S (3) Veale DJ (2), Fearon U (1)

Department(s)/Institutions

(1) Molecular Rheumatology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Ireland (2) Centre for Arthritis & Rheumatic Diseases, Dublin Academic Medical Centre, University College Dublin, Ireland (3) Immunology, Janssen Research & Development, 1400 McKean Road, Spring House, PA 19477, USA;

Introduction

Monocytes and macrophages play a key role in RA disease progression, however, the diversity and plasticity of cell subsets and their metabolic profile in inflammatory arthritis has yet to be elucidated.

Aims/Background

To elucidate the inflammatory and metabolic profiles/function of RA monocytes and macrophages and fully characterise RA synovial tissue macrophage subsets.

Method

CD14+ monocytes from RA, Arthralgia and HC bloods were isolated and examined ex-vivo or following differentiation into M1/M2 macrophages. Inflammatory mediators, metabolic markers and transcriptional activity was determined by RT-PCR and western blot, and phagocytosis capacity by OVA-albumin assays. Glycolysis (ECAR) and oxidative phosphorylation (OCR) were quantified by Seahorse-XFE-technology. Single cell synovial-tissue suspensions from RA,PsA and OA biopsies and synovial fluid cells were analysed for macrophage subsets (CD40,-CD45,-CD64,-CD68,-CD163,-CD206,-CD253) by flow cytometry. Double positive CD206+CD163+ vs CD206-CD163- cells were sorted by FACSAria Fusion sorter and RNAseq transcriptomic analysis performed.

Results

RA monocytes are hyperinflammatory, with significantly higher expression of IL-1β,TNFα,IL-6,OSM,CXCL10 and CXCL11 compared to HC (all p<0.05), this profile is maintained in monocyte-derived M1-macrophages and M2 macrophages with impaired phagocytic capacity observed. In parallel, an increase in HIF1α and PFKFB3 (a key glycolytic enzyme) was observed compared to HC (all-p<0.05). Baseline glycolysis (p<0.05), the maximal glycolytic capacity (p<0.05) and the ECAR:OCR ratio were increased in RA CD14+ monocytes and RA M1 macrophages compared to HC (p<0.05). Interestingly, this pro-hyperinflammatory/metabolic phenotype was also evident in CD14+ monocytes from arthralgia ACPA+/RF+ subjects. Transcriptional activation of STAT3 induced this hyperinflammatory/metabolic phenotype, with STAT3-inhibition resulting in metabolic reprogramming and resolution of inflammation. A significant enrichment of a dominant double positive CD206+CD163+ compared to CD206-CD163- macrophages was demonstrated in synovial-tissue versus fluid (p<0.05), with a significant increase in the frequency of CD206+CD163+CD40+ macrophages in RA synovial-tissue compared to PsA and OA (p<0.05). Finally, RNAseq revealed differential expression of genes involved in immune activation, cell adhesion and metabolism in CD206+CD163+ vs CD206-CD163- synovial tissue macrophages.

Conclusions

This study demonstrates a unique inflammatory and metabolic phenotype of RA monocytes/macrophages, a phenotype that may precede disease onset. Furthermore we have identified, for the first time, enrichment of a dominant transcriptionally distinct macrophage subtype in RA synovial-tissue.