Aging Rewires Neutrophils Across the Body, Single-Cell Map Reveals

Neutrophils have long been cast as the immune system’s expendable foot soldiers: short-lived, fast-acting cells that rush to sites of infection, engulf microbes, and die within hours. A new study published in Aging Cell suggests they may also be quiet architects of aging itself. By integrating single-cell RNA-sequencing data from eight tissues of young and aged mice, researchers constructed a unified atlas of nearly 257,000 cells and found that neutrophils do not merely accumulate in aging organs—they undergo a coordinated transcriptional transformation that appears to be conserved, at least in part, in humans.

The team, led by Yuman Wang and Teding Chang, assembled publicly available and newly generated datasets spanning the lung, heart, brain, vasculature, peripheral blood, intestine, kidney, and liver. After quality control and integration using reciprocal principal component analysis, the atlas comprised 66 samples and 256,949 cells. The compositional analysis revealed a striking pattern: neutrophil abundance rose markedly in several aged tissues. In the liver, the proportion of neutrophils among captured cells climbed from 0.75 percent in young mice to 5.26 percent in old ones; in the lung it rose from 0.88 to 4.14 percent; in vascular tissue from 1.05 to 3.68 percent; and in the intestine from 1.22 to 3.57 percent. The brain, shielded by the blood–brain barrier, showed no comparable increase.

These numbers were not artifacts of sequencing. Immunofluorescence staining for CD11b and myeloperoxidase confirmed dense neutrophil infiltration in aged lung, intestine, vasculature, and liver, while flow cytometry of bone marrow revealed a higher proportion of neutrophils in aged animals. A Bayesian compositional method called scCODA, which treats each biological sample as the unit of inference, independently supported a credible age-associated increase in neutrophil abundance in the liver, with a log2 fold change of 2.61 and an inclusion probability of 0.968.

Functionally, the aged neutrophils looked different at the molecular level. Pseudobulk differential expression analysis, which aggregates cells by sample to avoid inflating statistical significance, identified 26 genes upregulated in neutrophils from aged mice and 985 enriched in young ones. Gene set enrichment showed that glycolysis and reactive oxygen species pathways were activated in the aged cells, while TNF-α and interferon-λ signaling dominated in young ones. When the researchers isolated bone marrow neutrophils and cultured them for four hours, cells from aged mice died more readily and formed more neutrophil extracellular traps—web-like structures of DNA and antimicrobial proteins that, when deployed excessively, can damage surrounding tissue.

The most provocative experiments involved conditioned medium. When culture fluid from aged neutrophils was applied to mouse vascular smooth muscle cells and hepatocytes, both cell types showed increased beta-galactosidase activity, a classic marker of cellular senescence, along with elevated expression of matrix metalloproteinases and senescence-associated proteins such as P16 and P21. Quantitative PCR further revealed increased expression of senescance-associated secretory phenotype genes, including Il6, Tnf, and Ifn, in the exposed cells. In other words, factors released by aged neutrophils appeared to nudge tissue cells toward a stress-associated, inflammatory state.

To dissect neutrophil heterogeneity, the team re-clustered 5,210 neutrophils into 16 transcriptionally distinct subtypes, ranging from TNF-α-producing inflammatory cells to complement-associated, IL-6-expressing, E2F-driven, and unfolded-protein-response states. Several subtypes were preferentially enriched in aged mice, including Neu05-Complement, Neu06-IL6, and Neu08-E2F, while Neu01-TNFA and Neu04-Myc characterized young animals. Maturation scoring suggested that Neu05 and Neu08 resembled early marrow-emigrant neutrophils, mapping closest to the preNeu developmental stage, hinting that aged tissues may be receiving a wave of immature, hyper-responsive cells.

Cross-tissue co-occurrence analysis added a systems-level dimension. The researchers identified seven cellular modules whose member cell types varied in concert across samples. One module, CM3, was the most neutrophil-enriched and contained endothelial cells and fibroblasts alongside aged-enriched neutrophil states, suggesting that neutrophil accumulation covaries with stromal remodeling. CellChat-based ligand–receptor modeling predicted that aged endothelial cells, fibroblasts, and hepatocytes shift from CXCL12-dominant signaling toward CXCL1, CCL7, CCL6, and CCL4 signals. The young-enriched Neu01-TNFα subset preferentially engaged the CXCL12–CXCR4 axis, whereas the age-expanded Neu05 and Neu06 subsets were associated with CXCL1–CXCR2, providing a plausible chemotactic mechanism for the tissue-specific infiltration patterns.

The Neu06-IL6 state emerged as a central character. Pseudotime analysis placed these cells at the distal end of an inferred trajectory, with aged mice showing extended trajectory length and enrichment of Neu06 cells along the late path. The transcription factor CEBPB, which drives emergency granulopoiesis, and STAT3, the canonical IL-6 downstream effector, both increased along the aged ordering. In vitro, recombinant IL-6 activated STAT3 and raised reactive oxygen species production in HL-60 neutrophil-like cells, and conditioned medium from IL-6-stimulated cells induced senescence and matrix-remodeling markers in vascular smooth muscle and liver cell lines—an effect that persisted even after IL-6 washout, implying that IL-6-primed neutrophils release additional factors that propagate the signal.

The study also looked upstream, to the bone marrow. Single-cell analysis of hematopoietic progenitors revealed an aged-enriched state, HPC11, characterized by myeloid-biased genes such as Gata2, Car2, and Prtn3, in contrast to the lymphoid-primed HPC5 state enriched in young mice. Computational perturbation analysis predicted that FGF2 and angiopoietin-like 4 signaling shape the HPC11 program, consistent with the well-known myeloid skewing of aging hematopoiesis and providing a potential source for the flood of atypical neutrophils observed in peripheral tissues.

The authors are careful about causality. Their in vivo findings remain primarily associative, and the term aging-associated neutrophils refers to states enriched in aged organisms rather than neutrophils undergoing canonical senescence. Computational ligand–receptor and regulatory-network inferences are explicitly hypothesis-generating. Still, the partial conservation of these transcriptional programs in human multi-organ data from the Tabula Sapiens atlas, where older donors showed higher proportions of inflammatory and stress-responsive neutrophil states, lends weight to the framework. If future depletion, adoptive-transfer, or IL-6 blockade experiments confirm that these cells actively drive tissue decline, neutrophils may graduate from immune foot soldiers to prime suspects in the biology of aging—and targets for interventions aimed at slowing it.

Subject of Research: Aging-associated neutrophil remodeling across multiple mouse organs identified by single-cell transcriptomics

Article Title: Single‐Cell Mapping Identifies Aging‐Associated Neutrophil Remodeling Across Multiple Organs

Article References: Single‐Cell Mapping Identifies Aging‐Associated Neutrophil Remodeling Across Multiple Organs. (n.d.). https://doi.org/10.1111/acel.70735

Image Credits: AI Generated

DOI: 10.1111/acel.70735

Keywords: neutrophils, aging, single-cell RNA sequencing, IL-6, senescence, bone marrow, granulopoiesis, inflammation, NETs, chemokines, STAT3, geromedicine