Low-density granulocytes: functionally distinct, immature neutrophils in rheumatoid arthritis with altered properties and defective TNF signalling

Our aim was to determine whether rheumatoid arthritis (RA) low-density granulocytes (LDGs) are functionally different from RA neutrophils. LDGs from 32 RA patients were characterized using flow cytometry and quantitative PCR (qPCR). RNA sequencing (RNA-Seq) was carried out on paired RA LDGs and neutrophils (n = 4) and validated using qPCR. Functional assays included chemotaxis, phagocytosis, reactive oxygen species (ROS) production, cell-cycle analysis, apoptosis, neutrophil extracellular trap (NET)osis, and measurement of cytokine production (n >= 5 paired RA LDGs/neutrophils). RA LDGs had a substantially altered transcriptome, expressing >5000 genes at significantly different levels compared with RA neutrophils, including elevated levels of transcripts for granule proteins [including elastase and myeloperoxidase (MPO)] and cell-cycle genes [including cyclin-dependent kinase (CDK)2, CDK4, and CDK6]. Approximately 1% of RA LDGs stained positive for the G2/S phase of the cell cycle. RA LDGs had a significantly lower constitutive rate of apoptosis compared with RA neutrophils and did not respond to TNF-alpha in culture. Expression of transcripts for cytokines and cytokine receptors was lower in RA LDGs. NET formation was lower in LDGs in response to PMA compared with RA neutrophils. Chemotaxis and phagocytosis was lower in RA LDGs compared with neutrophils. RA LDGs produced significantly lower amounts of ROS in response to fMLP following priming with TNF-alpha. Expression of TNFR1 and -2 mRNA and protein was significantly lower in LDGs. We conclude that RA LDGS are functionally different from RA neutrophils, representing an immature neutrophil population within peripheral blood. Their enhanced survival properties and decreased TNF signaling are likely to have important consequences for disease pathology and response to therapy.