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

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.

Cytokine Networks in the Pathogenesis of Rheumatoid Arthritis

“…In RA patients, reactive oxygen species (ROS) are highly expressed in neutrophils and synovium [145]. RANKL itself induces nitric oxide synthase (NOS), and N-acetyl cysteine (NAC) inhibits RANKL-induced ROS production and differentiation of osteoclasts in bone marrow monocyte-macrophage lineage cells [146].…”

Mitochondria as Key Players in the Pathogenesis and Treatment of Rheumatoid Arthritis

“…Neutrophils also play a role in RA disease, contributing to inflammatory and destructive processes and acting as sources of autoantigens ( 57 ). Neutrophils rely heavily on glycolysis for energy production ( 58 ), and neutrophils from RA synovial fluid displayed an enhanced glycolytic gene signature compared with peripheral blood neutrophils from the same patients ( 57 ). Changes in mitochondrial activity have been shown to impact upon neutrophil functions such as chemotaxis ( 58 ), however this has not yet been demonstrated in the context of RA.…”

“…NETosis is a form of inducible cell death that occurs in neutrophils involving the release of complexes of decondensed chromatin and anti-microbial proteins, termed neutrophil extracellular traps (NETs). NETs function to trap and neutralise infectious agents, but are also implicated in the pathogenesis of autoimmune diseases including SLE and RA through stimulation of cytokine production and the release or extracellular generation of citrullinated proteins that act as autoantigens ( 57 , 61 , 195 – 197 ). Hypopolarised mitochondria have been shown to be released during NETosis, and ox-mtDNA contributes to NET formation in SLE, promoting interferon production and interferon stimulated gene signatures ( 61 ).…”

Low Density Neutrophils Are Increased in Patients With Behçet’s Disease but Do 3 Not Explain Differences in Neutrophil Function

“…Finally, as discussed above LDN are not a homogenous, stable population and further investigation of such diversity in BD is needed both in blood and tissue. (48) In summary, this study may indicate towards a dysfunctional phenotype and function displayed by neutrophils in BD. Taken together the data suggests that in BD persistent inflammation drive release of LDN from the bone marrow.…”