Lucy Gill

Neutrophil function declines with age, but little is known about the changes in neutrophil metabolism that take place in older people and people with frailty. We used ¹ H-NMR spectroscopy to investigate the metabolomic and lipidomic profiles of blood neutrophils from healthy younger (HY) and older (HO) people and older people with frailty (FR), to identify metabolites and metabolic pathways altered with healthy ageing and ageing with frailty. We also compared FR metabolites to rheumatoid arthritis (RA) neutrophils to identify markers of inflammation in FR neutrophils. We identified 17 significantly different polar metabolites across the participant groups (ANOVA adj. p<0.05). Three polar metabolites were significantly different in FR vs HO neutrophils, with NADP being higher in FR and lysine and choline being lower. NADP was uniquely elevated in FR compared to HO, HY and RA neutrophils. Seven metabolites were significantly different between HO and HY neutrophils, and only two metabolites were different between FR and RA neutrophils, identifying underlying similarities between FR and RA that may be driven by inflammation. Four lipid peaks were significantly different between FR and HY neutrophils. These were attributed to total cholesterol and omega 6 fatty acids. This is the first study of polar and lipid metabolism in neutrophils from people with frailty, providing evidence that metabolic signalling pathways involved in neutrophil activation, oxidative stress and lipid metabolism are altered during healthy ageing and ageing with frailty, and we identify NADP as a unique biomarker of altered neutrophil function in people with frailty.

Background/Objectives: Untargeted ¹H NMR metabolomics is a robust and reproducible approach used to study the metabolism in biological samples, providing unprecedented insight into altered cellular processes associated with human diseases. Metabolomics is increasingly used alongside other techniques to detect an instantaneous altered cellular function, for example, the role of neutrophils in the inflammatory response. However, in some clinical settings, blood samples may be limited, restricting the amount of cellular material available for a metabolomic analysis. In this study, we wanted to establish an optimal 1D ¹H NMR metabolomic pipeline for use with human neutrophil samples with low amounts of input material. Methods: We compared the effect of different neutrophil isolation protocols on metabolite profiles. We also compared the effect of the absolute cell counts (100,000 to 5,000,000) on the identities of metabolites that were detected with an increasing number of scans (NS) from 256 to 2048. Results/Conclusions: The variance in the neutrophil profile was equivalent between the isolation methods, and the choice of isolation method did not significantly alter the metabolite profile. The minimum number of cells required for the detection of neutrophil metabolites was 400,000 at an NS of 256 for the spectra acquired with a cryoprobe (700 MHz). Increasing the NS to 2048 increased metabolite detection at the very lowest cell counts (<400,000 neutrophils); however, this was associated with a significant increase in the analysis time, which would be rate-limiting for large studies. The application of a correlation-reliability-score-filtering method to the spectral bins preserved the essential discriminatory features of the PLS-DA models whilst improving the dataset robustness and analytical precision.

Tyrosine kinase inhibitors (TKIs) are widely used in cancer therapy yet are strongly associated with acute and chronic cardiotoxicity in patients. There is a critical need to advance our understanding of the pathophysiology that underlies TKI-mediated cardiotoxicity, and central to this is the use of reproducible and relevant preclinical models, which are employed in the evaluation of TKIs across the drug discovery pipeline. We have conducted a systematic review of the literature to determine how rodent models are used in the measurement of TKI-induced cardiotoxicity, focusing on animal reports, physiological cardiac outputs, histopathology, and biomarkers. A PRISMA-compliant systematic review was conducted using PubMed, Scopus, and Web of Science to identify studies reporting on TKI-induced cardiotoxicity in rodents. Only controlled in vivo, primary in vitro, and ex vivo studies using rats, mice, hamsters or guinea pigs were included. Data were extracted on species, strain, sex, age, experimental design, and cardiac outcomes with risk of bias analyses performed using the SYRCLE and SciRAP tools. Among 92 studies, sunitinib, imatinib, and sorafenib were the most frequently examined TKIs, with cardiotoxicity exhibited as altered cardiac functional parameters, fibrotic changes, arrhythmias, and elevated cardiac biomarkers. Rats (51 studies) and mice (46 studies) were predominantly used to study the effects of TKIs, whilst guinea pigs were underrepresented, limiting insights into electrophysiological changes that are associated with cardiotoxicity. Most studies used male rodents, and only two studies assessed age-related effects. Comparison between species strains was rarely conducted, despite evidence of this being a contributing factor to pre-disposition to cardiotoxicity. Rodent models were shown to replicate TKI-induced cardiotoxic effects observed in humans, but risk of bias analyses revealed limited evidence for study randomisation, inconsistent blinding, lack of sex-balanced studies, and poor strain diversity. Poor methodological quality and reporting across studies compromised reproducibility and interpretation of clinical relevance. Our study highlights the need for implementation of standardised protocols, strain, sex and age-stratified analyses to better support preclinical-to-clinical translation, as well as improve the safety of TKIs for patients and ensure more ethical use of animals in research.

Neutrophils contribute to systemic lupus erythematosus (SLE) pathogenesis through reactive oxygen species and neutrophil extracellular trap (NET) production, and increased apoptotic debris which causes autoantibody production and immune complex formation. These processes drive inflammation and tissue damage. The aim of this study was to perform integrated transcriptomic and metabolomic analyses comparing paediatric and adult SLE neutrophils. Adult (aSLE) and paediatric (jSLE) patient and healthy adult (HA) and juvenile (HJ) control neutrophils were subjected to RNAseq and 1H-NMR metabolomics. Univariate, multivariate and multiomics enrichment analyses were conducted in R and with ingenuity pathway analysis (IPA). Transcriptomic analysis revealed distinct gene expression profiles. Adult and juvenile SLE neutrophils were enriched for genes regulating interferon (IFN)-α/β signalling, neutrophil degranulation and NET signalling pathways (IPA, adj.P-value 1 metabolites were enriched in taurine/hypotaurine and amino acid metabolism in aSLE. Integrating transcriptomic and metabolomic data strengthened IFN-α/β signalling, neutrophil degranulation and NET signalling (adj. P < 0.001). Additional metabolic pathways uniquely down-regulated in aSLE included glutamate and glutamine metabolism, nucleotide biosynthesis and tryptophan catabolism (adj.P< 0.01). In summary, neutrophils from SLE patients, especially in jSLE, displayed complex transcriptomic and metabolic profiles, with aberrant IFN responses and neutrophil activation.