250 The effect of JAK inhibition on neutrophil killing, netosis and metabolism in rheumatoid arthritis

Extract

Background: JAK inhibitors (JAKi) tofacitinib and baricitinib are clinically effective in treating rheumatoid arthritis (RA). However, clinical trials of both drugs report increased infection rates and transient neutropenia during therapy. Neutrophils contribute to RA pathology via secretion of degradative enzymes, reactive oxygen species (ROS), cytokines and chemokines. However, neutrophils play a key role in host defence against micro-organisms and therefore impairment of normal neutrophil function by JAKi has serious implications for patients. The aim of this work was to determine the effect of JAKi on normal neutrophil functions relating to bacterial killing.

Methods: bNeutrophils were isolated from healthy controls (n = 10) and patients with RA (n = 10 DMARD-naïve, n = 10 Biologics-naïve) and pre-incubated with baricitinib, tofacitinib or a pan-JAK inhibitor (all 200ng/mL) for 30 min. ROS production was measured by DHR-123 or luminol-enhanced chemiluminescence; chemotaxis was measured by migration through hanging inserts; phagocytosis was measured using FITC-labelled latex beads; NETosis was measured by DNA assay and immuno-fluorescence. Neutrophil killing of S. aureus was also measured. For metabolomics, neutrophils were treated with JAKi for two hours before snap-freezing. Metabolites were extracted by 50:50 v/v AcN:H₂O and analysed by ¹H NMR spectroscopy using a 700 MHz Avance IIIHD Bruker NMR spectrometer equipped with a TCI cryoprobe. Chenomx, Bruker TopSpin and AMIX software were used to identify metabolites and process spectra. Statistical analysis was carried out using Metaboanalyst.