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

Abstract

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 30min. 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 700MHz 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. Results: JAKi did not affect chemotaxis or phagocytosis, with the exception of pan-JAK inhibition, which significantly impaired chemotaxis of RA neutrophils towards fMLP (p<0.01). JAKi significantly decreased the amount of H2O2-derived ROS produced by GM-CSFprimed neutrophils in response to fMLP stimulation (p<0.01). ROS production by unprimed neutrophils incubated with S. aureus was not significantly inhibited. Tofacitinib and baricitinib decreased the level of bacterial killing by around 5% from normal levels, and pan-JAK inhibition decreased the level of killing by around 10% (p<0.05). Whilst pan-JAK inhibition decreased NETosis in neutrophils stimulated with PMA, both baricitinib and tofacitinib increased NETosis. Levels of intra-cellular glucose, aspartate and homocysteine were significantly higher in neutrophils incubated with JAKi (FDR-adjusted p<0.05). Conclusion: JAK inhibition with tofacitinib, baricitinib or pan-JAK inhibitor I significantly decreased H2O2-derived, intracellular ROS production by primed neutrophils in response to fMLP, and decreased killing of S. aureus bacteria by around 5-10%. NET production in response to PMA was increased by baricitinib and tofacitinib. Changes in metabolism of glucose, aspartate and homocysteine may be a cause or consequence of these changes in functional activation. The observed changes in normal neutrophil function are likely to contribute to the increase in infections observed in patients receiving JAK inhibitors for the treatment of inflammatory diseases such as RA.