Bleomycin as an Inducer of Senescence in RAW 264.7 Macrophages

Bleomycin can induce macrophage senescence without triggering inflammatory activation, making it a useful tool for studying senescence in vitro.

Summary and findings

This study evaluated bleomycin as an inducer of senescence in RAW 264.7 murine macrophages, comparing it to LPS. Bleomycin resulted in 80% senescence-associated beta-galactosidase positivity and ~3-fold p53 protein accumulation, while LPS did not induce senescence markers. The findings suggest that bleomycin can effectively induce macrophage senescence independent of inflammatory activation.

Abstract

<title>Abstract</title> <p> Macrophage senescence is implicated in age-related immune dysfunction, but <italic>in vitro</italic> models remain limited, in part because the common use of lipopolysaccharide as an inducer also triggers acute inflammatory activation, making it difficult to attribute downstream phenotypes to senescence rather than to M1-type polarisation. Here, we evaluated bleomycin as an alternative senescence inducer in RAW 264.7 murine macrophages and compared it directly to LPS. Bleomycin produced a robust senescent phenotype: 80% senescence-associated beta-galactosidase (SA-β-gal) positivity, ~3-fold p53 protein accumulation with stable Trp53 transcript levels, strong Cdkn1a/p21 upregulation, characteristic morphological changes, and ~80% viability. Ki-67 expression was near-completely abolished, confirming proliferative arrest, with no transcriptional induction of the inflammatory cytokines Il6, Il1a, or Cxcl1 at 48 h. LPS produced the inverse: no SA-β-gal induction, no p21 upregulation, significant Trp53 downregulation, high nitric oxide and ROS output, massive upregulation of Il-6, IL-1α, and CXCL1, and viability below 30%. These findings identify bleomycin as a reliable inducer of macrophage senescence that is cleanly separable from inflammatory activation, and show that the two programs diverge not only on senescence markers but also on inflammatory gene transcription. This model provides a tractable system for studying the cell-autonomous consequences of the senescent state in macrophages independently of the confounding M1 response. </p>

Elsewhere in the P21 (P021) corpus