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 analysed 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 signalling 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, including exposure of myeloperoxidase (MPO) and citrullinated histone-H3-positive DNA NETs. IPA analysis predicted NET production was mediated by signalling 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.

Neutrophils in the Pathogenesis of Rheumatoid Arthritis and Systemic Lupus Erythematosus: Same Foe Different M.O.

“…Apoptosis is delayed in both blood and synovial fluid neutrophils from patients with RA ( 48 , 49 ). The likely cause of this is inflammatory cytokines, such as GM-CSF, TNFα, IL1β, and interferons, elevated in both blood and synovial fluid ( 50 ), which have been demonstrated to delay neutrophil apoptosis in in vitro experiments ( 32 – 34 , 37 , 51 – 55 ).…”

“…Many neutrophil genes, including those coding for granule proteins, migratory proteins, chemokines, and chemokine receptors are also regulated by circadian rhythms ( 19 ). Dynamic changes in neutrophil gene expression also take place as cells migrate from the blood into inflamed tissues during inflammation, with transcripts for adhesion molecules decreasing and chemokine expression increasing at sites of inflammation ( 49 , 184 ). Polymorphisms within expression quantitative trait loci (eQTLs) have been found in over 160 genes which play a fundamental role in every stage of neutrophil biology, from granulopoiesis to activation during infection and ultimately apoptosis.…”

“…IFNα has recently been shown to regulate neutrophil activation in the presence of inflammatory cytokines ( 173 ) and in particular is involved in a switch in expression of chemokine genes. Whilst expression of genes for CCL and CXCL family chemokines is relatively low in blood neutrophils from RA patients with a high interferon gene expression signature, the expression of these chemokines increases significantly in RA synovial fluid neutrophils following migration to inflamed joints ( 49 , 189 – 191 ). RA blood neutrophils also express mRNA for MHC Class II, with both RNA and MHC Class II protein being detected in RA synovial fluid neutrophils ( 192 ).…”

Identification of the difference of neutrophils in different locations in RA patients

“…Neutrophils are the common cell type in RA synovial uid and are also detected in RA synovial tissues 14 .…”

Crosstalk between B cells and neutrophils in rheumatoid arthritis

“…Intriguingly, patients with RA, as well as rats in an RA model, were found to harbour circulating low‐density granulocytes, with differential cell surface marker and gene expression signatures that are the subject of ongoing investigation [31–33]. SF neutrophils were reported to be more activated still and having a differential gene expression signature compared to circulating cells from the same patient [34]. SF neutrophils are longer‐lived than circulating neutrophils [35,36], secrete proteases, and release cytokines and chemokines to activate and recruit further neutrophils [29,37].…”