Neutrophil Extracellular Traps (NETs) are implicated in the development of auto-immunity in diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) through the externalization of intracellular neoepitopes e.g., dsDNA and nuclear proteins in SLE and citrullinated peptides in RA. The aim of this work was to use quantitative proteomics to identify and measure NET proteins produced by neutrophils from healthy controls, and from patients with RA and SLE to determine if NETs can be differentially-generated to expose different sets of neoepitopes. Ultra-pure neutrophils (>99%) from healthy individuals (n = 3) and patients with RA or SLE (n = 6 each) were incubated ± PMA (50 nM, PKC super-activator) or A23187 (3.8 μM, calcium ionophore) for 4 h. NETs were liberated by nuclease digestion and concentrated onto Strataclean beads prior to on-bead digestion with trypsin. Data-dependent LC-MS/MS analyses were conducted on a QExactive HF quadrupole-Orbitrap mass spectrometer, and label-free protein quantification was carried out using Progenesis QI. PMA-induced NETs were decorated with annexins, azurocidin and histone H3, whereas A23187-induced NETs were decorated with granule proteins including CAMP/LL37, CRISP3, lipocalin and MMP8, histones H1.0, H1.4, and H1.5, interleukin-8, protein-arginine deiminase-4 (PADI4), and α-enolase. Four proteins were significantly different between PMA-NETs from RA and SLE neutrophils (p < 0.05): RNASE2 was higher in RA, whereas MPO, leukocyte elastase inhibitor and thymidine phosphorylase were higher in SLE. For A23187-NETs, six NET proteins were higher in RA (p < 0.05), including CAMP/LL37, CRISP3, interleukin-8, MMP8; Thirteen proteins were higher in SLE, including histones H1.0, H2B, and H4. This work provides the first, direct comparison of NOX2-dependent (PMA) and NOX2-independent (A23187) NETs using quantitative proteomics, and the first direct comparison of RA and SLE NETs using quantitative proteomics. We show that it is the nature of the stimulant rather than neutrophil physiology that determines NET protein profiles in disease, since stimulation of NETosis in either a NOX2-dependent or a NOX2-independent manner generates broadly similar NET proteins irrespective of the disease background. We also use our proteomics pipeline to identify an extensive range of post-translationally modified proteins in RA and SLE, including histones and granule proteins, many of which are known targets of auto-antibodies in each disease.

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&gt;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&lt;0.05). RA SF significantly increased neutrophil migration through 3μM transwell chambers (p&lt;0.05) and also increased production of NETs by healthy control neutrophils (p&lt;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.

Pediatric Early Warning Scores are advocated to assist health professionals to identify early signs of serious illness or deterioration in hospitalized children. Scores are derived from the weighting applied to recorded vital signs and clinical observations reflecting deviation from a predetermined “norm.” Higher aggregate scores trigger an escalation in care aimed at preventing critical deterioration. Process errors made while recording these data, including plotting or calculation errors, have the potential to impede the reliability of the score. To test this hypothesis, we conducted a controlled study of documentation using five clinical vignettes. We measured the accuracy of vital sign recording, score calculation, and time taken to complete documentation using a handheld electronic physiological surveillance system, VitalPAC Pediatric, compared with traditional paper-based charts. We explored the user acceptability of both methods using a Web-based survey. Twenty-three staff participated in the controlled study. The electronic physiological surveillance system improved the accuracy of vital sign recording, 98.5% versus 85.6%, P < .02, Pediatric Early Warning Score calculation, 94.6% versus 55.7%, P < .02, and saved time, 68 versus 98 seconds, compared with paper-based documentation, P < .002. Twenty-nine staff completed the Web-based survey. They perceived that the electronic physiological surveillance system offered safety benefits by reducing human error while providing instant visibility of recorded data to the entire clinical team.