Single-cell immune profiling of mouse liver aging reveals Cxcl2+ macrophages recruit neutrophils to aggravate liver injury.

BACKGROUND AND AIMS: Immune cells play a crucial role in liver aging. However, the impact of dynamic changes in the local immune microenvironment on age-related liver injury remains poorly understood. We aimed to characterize intrahepatic immune cells at different ages to investigate key mechanisms associated with liver aging. APPROACH AND RESULTS: We carried out single-cell RNA sequencing on mouse liver tissues at 4 different ages, namely, the newborn, suckling, young, and aged stages. The transcriptomic landscape, cellular classification, and intercellular communication were analyzed. We confirmed the findings by multiplex immunofluorescence staining, flow cytometry, in vitro functional experiments, and chimeric animal models. Nine subsets of 89,542 immune cells with unique properties were identified, of which Cxcl2+ macrophages within the monocyte/macrophage subset were preferentially enriched in the aged liver. Cxcl2+ macrophages presented a senescence-associated secretory phenotype and recruited neutrophils to the aged liver through the CXCL2-CXCR2 axis. Through the secretion of IL-1ß and TNF-α, Cxcl2+ macrophages stimulated neutrophil extracellular traps formation. Targeting the CXCL2-CXCR2 axis limited the neutrophils migration toward the liver and attenuated age-related liver injury. Moreover, the relationship between Cxcl2+ macrophages and neutrophils in age-related liver injury was further validated by human liver transplantation samples. CONCLUSIONS: This in-depth study illustrates that the mechanism of Cxcl2+ macrophage-driven neutrophil activation involves the CXCL2-CXCR2 axis and provides a potential therapeutic strategy for age-related liver injury.

Machine learning-based identification of CYBB and FCAR as potential neutrophil extracellular trap-related treatment targets in sepsis.

Objective: Sepsis related injury has gradually become the main cause of death in non-cardiac patients in intensive care units, but the underlying pathological and physiological mechanisms remain unclear. The Third International Consensus Definitions for Sepsis and Septic Shock (SEPSIS-3) definition emphasized organ dysfunction caused by infection. Neutrophil extracellular traps (NETs) can cause inflammation and have key roles in sepsis organ failure; however, the role of NETs-related genes in sepsis is unknown. Here, we sought to identify key NETs-related genes associate with sepsis. Methods: Datasets GSE65682 and GSE145227, including data from 770 patients with sepsis and 54 healthy controls, were downloaded from the GEO database and split into training and validation sets. Differentially expressed genes (DEGs) were identified and weighted gene co-expression network analysis (WGCNA) performed. A machine learning approach was applied to identify key genes, which were used to construct functional networks. Key genes associated with diagnosis and survival of sepsis were screened out. Finally, mouse and human blood samples were collected for RT-qPCR verification and flow cytometry analysis. Multiple organs injury, apoptosis and NETs expression were measured to evaluated effects of sulforaphane (SFN). Results: Analysis of the obtained DEGs and WGCNA screened a total of 3396 genes in 3 modules, and intersection of the results of both analyses with 69 NETs-related genes, screened out seven genes (S100A12, SLC22A4, FCAR, CYBB, PADI4, DNASE1, MMP9) using machine learning algorithms. Of these, CYBB and FCAR were independent predictors of poor survival in patients with sepsis. Administration of SFN significantly alleviated murine lung NETs expression and injury, accompanied by whole blood CYBB mRNA level. Conclusion: CYBB and FCAR may be reliable biomarkers of survival in patients with sepsis, as well as potential targets for sepsis treatment. SFN significantly alleviated NETs-related organs injury, suggesting the therapeutic potential by targeting CYBB in the future.

MSC-derived extracellular vesicles as nanotherapeutics for promoting aged liver regeneration.

Aging is one of the critical factors to impair liver regeneration leading to a high incidence of severe complications after hepatic surgery in the elderly population without any effective treatment for clinical administration. As cell-free nanotherapeutics, mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have been demonstrated the therapeutic potentials on liver diseases. However, the effects of MSC-EVs on the proliferation of aged hepatocytes are largely unclear. In this study, we found MSCs could reduce the expression of senescence-associated markers in the liver and stimulate its regeneration in aged mice after receiving a two-thirds partial hepatectomy (PHx) through their secreted MSC-EVs. Using RNA-Seq and AAV9 vector, we mechanistically found that these effects of UC-MSC-EVs partially attributed to inducing Atg4B-related mitophagy. This effect repairs the mitochondrial status and functions of aged hepatocytes to promote their proliferation. And protein mass spectrum analysis uncovered that DEAD-Box Helicase 5 (DDX5) enriches in UC-MSC-EVs, which interacts with E2F1 to facilitate its nuclear translocation for activating the expression of Atg4B. Collectively, our data show that MSC-EVs act nanotherapeutic potentials in anti-senescence and promoting regeneration of aged liver by transferring DDX5 to regulate E2F1-Atg4B signaling pathway that induce mitophagy, which highlights the clinical application valuation of MSC-EVs for preventing severe complications in aged population receiving liver surgery.