Metabolic Profiling of Rheumatoid Arthritis Neutrophils Reveals Altered Energy Metabolism That Is Not Affected by JAK Inhibition

Abstract

Neutrophils play a key role in the pathophysiology of rheumatoid arthritis (RA) where release of ROS and proteases directly causes damage to joints and tissues. Neutrophil function can be modulated by Janus Kinase (JAK) inhibitor drugs, including tofacitinib and baricitinib, which are clinically effective treatments for RA. However, clinical trials have reported increased infection rates and transient neutropenia during therapy. The subtle differences in the mode of action, efficacy and safety of JAK inhibitors have been the primary research topic of many clinical trials and systematic reviews, to provide a more precise and targeted treatment to patients. The aim of this study was to determine both the differences in the metabolome of neutrophils from healthy controls and people with RA, and the effect of different JAK inhibitors on the metabolome of healthy and RA neutrophils. Isolated neutrophils from healthy controls (HC) (n = 6) and people with RA (n = 7) were incubated with baricitinib, tofacitinib or a pan-JAK inhibitor (all 200 ng/mL) for 2 h. Metabolites were extracted, and 1H nuclear magnetic resonance (NMR) was applied to study the metabolic changes. Multivariate analyses and machine learning models showed a divergent metabolic pattern in RA neutrophils compared to HC at 0 h (F1 score = 86.7%) driven by energy metabolites (ATP, ADP, GTP and glucose). No difference was observed in the neutrophil metabolome when treated with JAK inhibitors. However, JAK inhibitors significantly inhibited ROS production and baricitinib decreased NET production (p < 0.05). Bacterial killing was not impaired by JAK inhibitors, indicating that the effect of JAK inhibitors on neutrophils can inhibit joint damage in RA without impairing host defence. This study highlights altered energy metabolism in RA neutrophils which may explain the cause of their dysregulation in inflammatory disease.

PFKFB3 is a critical regulator of neutrophil metabolism and function in rheumatoid arthritis

“…In our previous study we identified that the metabolome in RA neutrophils is different from HC, mainly driven by metabolites of central carbon metabolism (glucose, ATP, NADP, lactate) [18]. In this study we wanted to further investigate the reasons for this, and therefore we first analysed the expression of genes regulating glycolysis in RA neutrophils.…”

“…In this study we investigated HC and RA neutrophil responses when challenged with inhibitors of central carbon metabolism or low extracellular glucose concentrations. Recent studies have highlighted the importance of pathways connected to glycolysis in neutrophils, including glycogenolysis, gluconeogenesis, glutaminolysis and PPP [17, 18] and this work provides new information about the critical role of glucose metabolism in human neutrophils.…”

“…Neutrophils were prepared for metabolite extraction as previously described [18]. Briefly, cells were centrifuged at 1000g at 25 o C for 2 min, pellet was washed with ice cold PBS then centrifuged at 1000g at 25 o C for 2 min.…”

“…Compared to other immune cells, the study of neutrophil immunometabolism is still in the early discovery phase. We recently measured the neutrophil metabolome in RA using 1 H NMR metabolomics and identified that levels of intracellular glucose, lactate and NADP+ are different in RA neutrophils compared to healthy controls [18]. The reliance on glycolysis for cellular activation is a challenge for neutrophils as sites of inflammation such as the RA synovial joint are frequently characterised by limited nutrient availability, including glucose, as well as hypoxia [19].…”

Development of ADS051, an oral, gut‐restricted, small molecule neutrophil modulator for the treatment of neutrophil‐mediated inflammatory diseases

“…Ozanimod is FDA‐approved for moderately to severely active UC [ 8 ]. Tofacitinib is FDA‐approved for the treatment of RA, UC, and psoriatic arthritis [ 2 , 7 , 10 ]. Multiple clinical trials have investigated neutrophil‐targeting agents, including tofacitinib, in the context of several diseases and have found that treatment side effects exist [ 2 , 7 , 8 ].…”