Integrated transcriptomic and metabolomic analysis to reveal the mechanism of ischemic tolerance in TIA patients

Background: Transient ischemic attack (TIA) induces ischemic tolerance that can reduce the subsequent ischemic damage and improve prognosis of stroke patients. However, the underlying mechanisms remain elusive. Recent advances in plasma metabolomics analysis have made it a powerful tool to investigate human pathophysiological phenotypes and mechanisms of diseases. In this study, we aimed to identify the bioactive metabolites from the plasma of TIA patients for determination of their prophylactic and therapeutic effects on protection against cerebral ischemic stroke, and the mechanism of TIA-induced ischemic tolerance against subsequent stroke. Methods: Metabolomic profiling using liquid chromatography-mass spectrometry was performed to identify the TIA-induced differential bioactive metabolites in the plasma samples of 20 patients at day 1 (time for basal metabolites) and day 7 (time for established chronic ischemic tolerance-associated metabolites) after onset of TIA. Mouse MCAO-induced stroke model was used to verify their prophylactic and therapeutic potentials. Transcriptomics changes in circulating neutrophils of TIA patients were determined by RNA-sequencing. Multivariate statistics and integrative analysis of metabolomics and transcriptomics were performed to elucidate the potential mechanism of TIA-induced ischemic tolerance. Findings: Plasma metabolomics analysis identified five differentially upregulated metabolites associated with TIA-induced ischemic tolerance, namely all-trans 13,14 dihydroretinol (atDR), 20-carboxyleukotriene B4, prostaglandin B2, cortisol and 9-KODE. They were associated with the metabolic pathways of retinol, arachidonic acid, and neuroactive ligand-receptor interaction. Prophylactic treatment of MCAO mice with these five metabolites significantly improved neurological functions. Additionally, post-stroke treatment with atDR or 9-KODE significantly reduced the cerebral infarct size and enhanced sensorimotor functions, demonstrating the therapeutic potential of these bioactive metabolites. Mechanistically, we found in TIA patients that these metabolites were positively correlated with circulating neutrophil counts. Integrative analysis of plasma metabolomics and neutrophil transcriptomics further revealed that TIA-induced metabolites are significantly correlated with specific gene expression in circulating neutrophils which showed prominent enrichment in FoxO signaling pathway and upregulation of the anti-inflammatory cytokine IL-10. Finally, we demonstrated that the protective effect of atDR-pretreatment on MCAO mice was abolished when circulating neutrophils were depleted. Interpretation: TIA-induced ischemic tolerance is associated with upregulation of plasma bioactive metabolites which can protect against cerebral ischemic damage and improve neurological functions through a positive role of circulating neutrophils. Overall design: To elucidate the potential mechanism of TIA-induced ischemic tolerance, we performed transcriptomic analyses in the circulatory neutrophils as well as metabolomic analyses in the plasma samples collected from 20 TIA patients at day 1 and day 7 after their TIA onset. We performed gene expression profiling analysis using data obtained from RNA-sequencing of the two time points. Integrative analyses of the transcriptomic and metabolomic data were then conducted.