Comprehensive single-cell analysis deciphered the immunoregulatory mechanism of TPPU in alleviating sepsis-related acute liver injury

Introduction: Sepsis-related acute liver injury involves complex immune dysfunctions. Epoxyeicosatrienoic acids (EETs), bioactive molecules derived from arachidonic acid (AA) via cytochrome P450 (CYP450) and rapidly hydrolyzed by soluble epoxide hydrolase (sEH), possess anti-inflammatory properties. Nevertheless, the impact of the sEH inhibitor TPPU on sepsis-related acute liver injury remains uncertain. Objectives: This study utilized comprehensive single-cell analysis to investigate the immunoregulatory mechanism of TPPU in alleviating sepsis-related acute liver injury. Methods: Hepatic bulk RNA sequencing and proteomics analyses were employed to investigate the mechanisms underlying sepsis-related acute liver injury induced by cecal ligation and puncture in mice. Cytometry by time-of-flight and single-cell RNA sequencing were conducted to thoroughly examine the immunoregulatory role of TPPU at single-cell resolution. Results: Downregulation of AA metabolism and the CYP450 pathway was observed during sepsis-related acute liver injury, and TPPU treatment reduced inflammatory cytokine production and mitigated sepsis-related hepatic inflammatory injury. Comprehensive single-cell analysis revealed that TPPU promotes the expansion of anti-inflammatory CD206+CD73+ M2-like macrophages and PDL1-CD39-CCR2+ neutrophils, reprogramming liver neutrophils to an anti-inflammatory CAMP+NGP+CD177+ phenotype. Additionally, TPPU inhibits the CCL6-CCR1 signaling mediated by M2-like macrophages and CAMP+NGP+CD177+ neutrophils, altering intercellular communication within the septic liver immune microenvironment. Conclusion: This study demonstrated TPPU's protective efficacy against sepsis-related acute liver injury, underscoring its vital role in modulating liver macrophages and neutrophils and enhancing prospects for personalized immunomodulatory therapy. Overall design: CLP mice (CLP group) and sham mice (Sham group) were used to investigate liver transcriptome and proteome alterations during sepsis-related acute liver injury. To evaluate TPPU's effects on sepsis-related acute liver injury, CLP mice were randomly assigned to two groups: a control group (Ctrl group) receiving 0.9% sterile saline via oral gavage and a treatment group (TPPU group) receiving TPPU (3 mg/kg per day) orally for three consecutive days before CLP. Mice were euthanized for analysis 24 hours after CLP. A cytometry by time-of-flight (CyTOF) panel consisting of 42 metal-labeled antibodies was developed for this study. Single-cell sequencing (scRNA-seq) libraries were prepared as per the manufacturer's instructions. CyTOF and scRNA-seq were conducted to thoroughly examine the immunoregulatory role of TPPU at single-cell resolution.