# Ceramide Complex Ameliorates Metabolically Driven Neutrophil Senescence by Regulating Apoptosis via the cGAS-STING Pathway

## TL;DR

This study shows that ceramide can reduce aging-related neutrophil dysfunction by regulating apoptosis through the cGAS-STING pathway.

## Contribution

The novel finding is that ceramide ameliorates neutrophil senescence by modulating mitochondrial permeability and the cGAS-STING pathway.

## Key findings

- Aged mice show delayed apoptosis in neutrophils and reduced functionality.
- Ceramide intervention restores neutrophil function and promotes apoptosis.
- Ceramide modulates mitochondrial permeability and activates the cGAS-STING pathway.

## Abstract

Background: Population aging is increasingly recognized as a major global challenge. Researchers have identified a correlation between aging and immunosenescence, leading to dysfunction of the immune system. As a crucial component of the innate immune system, age-related changes in neutrophils have garnered significant attention from researchers, but the underlying mechanisms remain unclear. This study aims to comprehensively evaluate the senescence status and potential mechanisms of neutrophils, and to identify targets for delaying or even reversing senescence.

Methods: Blood routine tests and Luminex Multiplex Cytokine Analysis were employed to assess inflammation levels in mice. Flow cytometry and an agarose chemotaxis model were used to evaluate baseline biological functions and stress responses of neutrophils. Transmission electron microscopy and flow cytometry were utilized to compare mitochondrial ultrastructure and function. Metabolomic analysis was performed to examine metabolic patterns. qPCR, Western blotting, and flow cytometry were used to investigate the potential mechanisms of ceramide intervention on neutrophils.

Results: Our findings indicate that aged mice exhibit considerable variability in delayed apoptosis among bone marrow neutrophils, alongside a notable reduction in baseline functionality and stress response capabilities. Metabolomic analysis revealed a marked decrease in ceramide levels within aged neutrophils. In vitro ceramide intervention revitalized neutrophil functionality and partially inhibited delayed apoptosis, facilitating the efficient elimination of senescent neutrophils. The underlying mechanism behind these effects might be attributed to ceramide's modulation of mitochondrial permeability, which in turn influences the activation of the cGAS-STING pathway, as well as its regulatory role in maintaining the equilibrium of pro-apoptotic Bcl-2 protein levels.

Conclusions: This investigation proficiently assessed neutrophil senescence in terms of both biological functionalities and intrinsic diversity, while concurrently exploring the feasibility and primary mechanisms through which ceramide intervention impacts neutrophil senescence at the levels of signaling pathways, protein expression, and cellular microarchitecture. These findings provide novel insights into evaluating and potentially intervening in immune senescence, with implications for organismal aging.