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.

Keywords: neutrophil extracellular traps; neutrophils; rheumatoid arthritis; synovial fluid; transcriptomics.

Figure 1

Transcriptomic analysis of rheumatoid arthritis peripheral blood and synovial fluid (SF) neutrophils (PMN). (A) PLS-DA modeling of blood and SF neutrophil transcriptomes using mixOmics. Blood and SF neutrophils were discriminated based on two components with an AUC of 1.0 (p<0.05). (B) Modular analysis of gene expression networks in blood and SF neutrophils using tmod (AUC>0.8).

Figure 2

Transcriptomic analysis of genes differently expressed (1.5-fold change) between peripheral blood (PB) and synovial fluid (SF) neutrophils. (A) IPA signaling pathways predicted to be up-regulated (red) or down-regulated (green) in SF neutrophils. (B) Heatmap showing chemokine gene expression in PB and SF neutrophils. (C) Leukocyte extravasation pathway (p=1.41x10^-7) showing genes up-regulated (red) or down-regulated (green) in SF neutrophils (white = not expressed).

Figure 3

Activation of gene networks in RA SF neutrophils that control migration and activation of antigen presenting cells. These networks are regulated by (A) ERK, (B) MYD88, and (C) TICAM1.

Figure 4

Effect of RA SF on neutrophil migration and apoptosis. (A) Neutrophil chemotaxis was significantly increased towards fMLP, interleukin-8 (IL-8) and 10% RA SF (p < 0.0001). (B) Neutrophil apoptosis was significantly delayed by RA SF over 18 h (p < 0.05). (C) IPA apoptosis signaling pathway showing up-regulation (red) and down-regulation (green) of genes associated with regulation of apoptosis in SF neutrophils (white = not expressed).

Figure 5

Cytokines in RA SF prime the neutrophil respiratory burst. (A) IPA predicted up-stream cytokine regulators of gene expression in RA SF neutrophils (IFNG, interferon-γ; GCSF, granulocyte colony stimulating factor; TNF, tumor necrosis factor α; IL1B, interleukin 1β; IL6, interleukin 6). (B) Levels of cytokines measured in RA SF from which the neutrophils sequenced by RNA-seq were isolated. (C) ROS production by RA blood and SF neutrophils (with and without cytokine priming) to fMLP. RA SF (25% v/v) also activates ROS production in healthy neutrophils. Stimulation of neutrophils with RA SF containing soluble immune complexes (D) activates ROS production in cytokine primed neutrophils only, whereas RA SF containing insoluble immune complexes (E) activates ROS production in both primed and unprimed neutrophils.

Figure 6

Activation of NET production by RA SF. (A) IPA predicted activation of signaling cascades regulated by AKT, RAF1 and SRC which may regulate NET production. (B) Neutrophils were incubated for 4 h with PMA (50 nM), A23187 (3.8 uM), or 10% RA SF. NET production was increased by RA SF as measured by quantification of extracellular DNA in culture supernatants, DAPI staining of extracellular NET DNA (blue), and immunofluorescent staining for myeloperoxidase (MPO, red) and citrullinated histone H3 (cit-H3, green) on NET structures (*p < 0.05, **p < 0.01, ***p < 0.001). White arrows indicate examples of NETs on each panel.

Figure 7

Summary of the role of inflammatory role of chemokines expressed by RA SF neutrophils.