The short-acting GABA (A) receptor agonist remimazolam is an inhibitor of T cell activation and can induce phenotype change of regulatory T cells

Background: GABAA receptor genes are targets for intravenous anesthetics, but their expressions have been observed in multiple lineages of immune cells including T cells. However, knowledge of their agonists on T cells remains very limited, and unbiased system level characterizations of T cells in vivo following GABAA receptor agonist treatment have not been reported. Methods: The RNA Sequencing, qRT-PCR, CRISPR/Cas9 gene knockout, cell sorting, single cell sequencing and flow cytometry were used. Results: Remimazolam treatment significantly inhibited CD69 expression, ERK phosphorylation following T cell activation, and induced transcriptomic change in Jurkat T cells, such as TGFBI. Knockout of TGFBI significantly altered the inhibition of T cell activation by remimazolam. To assess impact of remimazolam, we first intraperitoneally injected C57BL/6 mice with remimazolam, sorted T cells and performed single-cell RNA sequencing (scRNA-seq) analyses. Single-cell RNA sequencing (scRNA-seq) analysis found that intraperitoneally injection of remimazolam into C57BL/6 mice resulted in significant changes in a Foxp3 expressing sub-cluster among T cells, even though differences in surface expression of T cell activation markers was not found by flow cytometric analyses, which was probably due to pharmacokinetic properties of the ultra-fast acting drug. Conclusions: Our experimental data have revealed the impact of GABAA receptor agonist on T cells and confirmed inhibitory role of remimazolam on T cell activation, which could provide insights into neuroimmunological links during anesthetic treatment. C57BL/6 female mice were divided into three groups of three mice each injected with saline, 6 mg/kg remimazolam for 4 h or 8 h, respectively. Then the spleens of mice were collected and gently dissociated in RPMI 1640 medium containing 10% FBS on ice using 1 ml syringe plunger on 100 μm cell strainer (BD), all samples were transferred into 15 mL tubes, respectively. Cell samples from spleens were stained with antibody mixture and sorted for single cell sequencing.