Rheumatoid Arthritis Synovial Fluid Neutrophils Drive Inflammation Through Production of Chemokines, Reactive Oxygen Species, and Neutrophil Extracellular Traps.

Max Lyon,Steven W Edwards,Robert J Moots,Helen L Wright,Elinor A Chapman

doi:10.3389/fimmu.2020.584116

Abstract

Rheumatoid arthritis (RA) is a chronic inflammatory disorder affecting synovial joints. Neutrophils are believed to play an important role in both the initiation and progression of RA, and large numbers of activated neutrophils are found within both synovial fluid (SF) and synovial tissue from RA joints. In this study we analyzed paired blood and SF neutrophils from patients with severe, active RA (DAS28>5.1, n=3) using RNA-seq. 772 genes were significantly different between blood and SF neutrophils. IPA analysis predicted that SF neutrophils had increased expression of chemokines and ROS production, delayed apoptosis, and activation of signaling cascades regulating the production of NETs. This activated phenotype was confirmed experimentally by incubating healthy control neutrophils in cell-free RA SF, which was able to delay apoptosis and induce ROS production in both unprimed and TNFα primed neutrophils (p<0.05). RA SF significantly increased neutrophil migration through 3μM transwell chambers (p<0.05) and also increased production of NETs by healthy control neutrophils (p<0.001), including exposure of myeloperoxidase (MPO) and citrullinated histone-H3-positive DNA NETs. IPA analysis predicted NET production was mediated by signaling networks including AKT, RAF1, SRC, and NF-κB. Our results expand the understanding of the molecular changes that take place in the neutrophil transcriptome during migration into inflamed joints in RA, and the altered phenotype in RA SF neutrophils. Specifically, RA SF neutrophils lose their migratory properties, residing within the joint to generate signals that promote joint damage, as well as inflammation via recruitment and activation of both innate and adaptive immune cells. We propose that this activated SF neutrophil phenotype contributes to the chronic inflammation and progressive damage to cartilage and bone observed in patients with RA.

Highlights

Rheumatoid arthritis (RA) is an inflammatory disorder characterized by systemic inflammation, including swelling and pain in synovial joints
In this study we have described for the first time the changes in gene expression that take place in RA neutrophils following migration from peripheral blood into inflamed joints
Using RNA-seq we have revealed an activated neutrophil phenotype in RA synovial fluid (SF), characterized by increased expression of chemokines, delayed apoptosis and increased activation of kinases and transcription factors which may be implicated in the production of neutrophil extracellular traps (NETs)

Summary

Introduction

Rheumatoid arthritis (RA) is an inflammatory disorder characterized by systemic inflammation, including swelling and pain in synovial joints. As well as having an activated phenotype in peripheral blood, activated neutrophils are found at high numbers in both synovial joints and tissues of patients with RA [5,6,7] Their presence within RA joints is accompanied by high levels of neutrophil granule proteins in synovial fluid, including myeloperoxidase (MPO), cathepsin G, proteinase 3, elastase, and lactoferrin [1, 8,9,10,11,12]. Ex vivo synovial fluid (SF) neutrophils have an altered phenotype compared to paired blood neutrophils They produce higher levels of superoxide (O·2−) and contain phosphorylated p47phox, indicating assembly and activation of the NADPH oxidase (NOX2) in vivo [17]. It was recently shown that up to 70% of newly-diagnosed RA patients have auto-antibodies in their serum that recognize NET components (ANETA) [28]

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