Aging Drives Neutrophil Buildup Across Multiple Organs, Fueling Inflammation
Single-cell mapping reveals how neutrophils accumulate and shift to inflammatory states in aged organs, with IL-6 driving tissue senescence.
Summary
Using single-cell RNA sequencing integrated across eight organs from young and aged mice, researchers identified a striking age-associated rise in neutrophil abundance — particularly in the liver, lung, vasculature, and intestine. Aged neutrophils displayed enhanced glycolysis and reactive oxygen species programs, formed more neutrophil extracellular traps, and secreted factors that triggered senescence in vascular smooth muscle and liver cells. A specific age-enriched neutrophil subset (Neu06-IL6) was characterized, with IL-6 signaling nominated as a key regulatory axis. Bone marrow analysis revealed an old-enriched progenitor population (HPC11) with enhanced myeloid bias, suggesting the aging inflammatory neutrophil phenotype originates upstream in hematopoietic stem cell programming. FGF signaling was computationally identified as a candidate regulator of these bone marrow shifts.
Detailed Summary
Neutrophils — the most abundant circulating white blood cells — are increasingly recognized as contributors to sterile inflammation and tissue damage during aging, yet their organ-level remodeling has not been systematically characterized. This study addresses that gap by integrating single-cell RNA sequencing (scRNA-seq) datasets from eight tissues — lung, heart, brain, vasculature, peripheral blood, intestine, kidney, and liver — capturing 256,949 cells from 66 samples in young and aged mice. Reciprocal principal component analysis (RPCA) was used to harmonize batch effects across sequencing platforms, producing one of the most comprehensive multi-organ immune atlases in aging research to date.
Neutrophil proportions increased markedly with age across several peripheral organs. The most dramatic shifts were in the liver (0.75% young vs. 5.26% old), lung (0.88% vs. 4.14%), vasculature (1.05% vs. 3.68%), and intestine (1.22% vs. 3.57%). Bayesian compositional analysis (scCODA) confirmed a credible age-associated increase in liver neutrophils (inclusion probability = 0.968; log2 fold change = 2.61). Immunofluorescence staining for CD11b and MPO validated neutrophil accumulation across these organs in vivo, and flow cytometry of bone marrow confirmed a higher neutrophil proportion in aged mice (p < 0.05). Notably, the brain showed no significant neutrophil increase, consistent with blood–brain barrier restriction of circulating immune cells.
A neutrophil-specific atlas of 5,210 cells (2,400 young, 2,810 old) was constructed and subjected to pseudobulk differential expression analysis. Twenty-six genes were upregulated and 985 genes were enriched in young neutrophils relative to aged ones. Gene set enrichment analysis showed glycolysis and reactive oxygen species (ROS) pathways were enriched in aged neutrophils, while TNF-α and IFN-λ signaling predominated in young neutrophils. Aged neutrophils also showed elevated NET formation by Cytox Green staining (p < 0.01). Conditioned media from aged mouse neutrophils applied to vascular smooth muscle cells (MOVAS) and hepatocyte-derived cells (AML-12) significantly increased senescence-associated β-galactosidase positivity, elevated MMP9, MMP12, P16, and P21 protein expression, and upregulated SASP genes including Il6, Tnf, and Mmp9 (all p < 0.05), demonstrating functional paracrine senescence induction.
Unsupervised clustering identified seven neutrophil subsets (Neu01–Neu07). One cluster, Neu06-IL6, was specifically enriched in aged mice and characterized by elevated expression of Il6, complement genes, and oxidative stress programs. In vitro stimulation of HL-60 neutrophil-like cells with IL-6 recapitulated inflammatory and senescence-associated transcriptional changes in co-cultured MOVAS and AML-12 cells, supporting IL-6 as a functional mediator of this aged neutrophil state. Spatial and communication analyses using CellChat further identified IL-6 as a prominent signaling axis between aged neutrophils and stromal cell populations across tissues.
Bone marrow scRNA-seq analysis of hematopoietic progenitor cells revealed an old-enriched population, HPC11, characterized by enhanced myeloid priming transcriptional signatures, suggesting that the upstream origin of inflammatory neutrophil states lies in progenitor-level reprogramming during aging. In silico perturbation analysis using CellOracle nominated FGF signaling as a candidate regulator of HPC subtype-specific transcriptional programs. Together, these findings construct a multi-organ framework for understanding how neutrophil remodeling — beginning in the bone marrow and propagating to peripheral tissues — contributes to the sterile inflammatory tissue environments that characterize biological aging, with IL-6 and FGF signaling as potential therapeutic targets.
Key Findings
- Liver neutrophil proportion rose from 0.75% in young to 5.26% in aged mice; lung from 0.88% to 4.14%; vasculature from 1.05% to 3.68%; intestine from 1.22% to 3.57%
- Bayesian scCODA confirmed credible age-associated liver neutrophil increase (inclusion probability = 0.968; log2 fold change = 2.61)
- Aged neutrophil atlas (5,210 cells) showed 26 genes upregulated with age; glycolysis and ROS pathways enriched in old, TNF-α and IFN-λ pathways enriched in young
- Conditioned media from aged neutrophils significantly increased senescence markers (P16, P21, MMP9, MMP12) and SASP gene expression (Il6, Tnf, Mmp9) in vascular and liver cells (p < 0.05)
- Aged neutrophil subset Neu06-IL6 showed elevated IL-6, complement, and oxidative stress gene programs; IL-6 stimulation of neutrophil-like cells in vitro reproduced paracrine senescence induction
- Bone marrow scRNA-seq identified old-enriched progenitor population HPC11 with enhanced myeloid priming, suggesting age-related neutrophil reprogramming begins at the stem cell level
- In silico perturbation analysis nominated FGF signaling as a candidate regulator of aging-associated hematopoietic progenitor transcriptional states
Methodology
The study integrated publicly available scRNA-seq datasets from eight organs (lung, heart, brain, vasculature, peripheral blood, intestine, kidney, liver) in young and aged mice, totaling 256,949 cells across 66 samples; RPCA integration was used to correct batch effects across sequencing platforms. Neutrophil-specific analyses included pseudobulk differential expression, gene set enrichment, unsupervised clustering into seven subsets, and Bayesian compositional analysis via scCODA. In vitro validation used conditioned media from neutrophils isolated from young and aged mice applied to MOVAS and AML-12 cell lines, with senescence quantified by β-galactosidase staining, immunoblotting, and qPCR. Statistical testing included Wilcoxon rank-sum tests, unpaired two-sided t-tests, and two-way ANOVA with Tukey correction; bone marrow flow cytometry used n = 6 mice per group.
Study Limitations
The study is primarily mouse-based, and while the organ-level findings are biologically compelling, direct translation to human aging biology requires validation in human cohorts. The in vitro conditioned-media experiments demonstrate paracrine effects but cannot fully recapitulate the complex in vivo tissue microenvironment; causal directionality between Neu06-IL6 neutrophils and tissue senescence remains to be established in vivo. The computational perturbation nominations for FGF signaling are hypothesis-generating only and require experimental validation. No conflicts of interest were declared by the authors.
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