Early-life exercise induces immunometabolic epigenetic modification enhancing anti-inflammatory immunity in middle-aged male mice

The study addresses whether early-life regular exercise confers long-term benefits to immune function and anti-inflammatory responses later in life. Prior longitudinal and retrospective human studies suggest that physical activity in youth improves adult health outcomes (aerobic fitness, bone density, lower BMI, reduced hypertension and type 2 diabetes). Exercise is known to modulate immune function and reduce chronic inflammation, but the durability of immune benefits from early-life exercise remains unclear. Emerging immunometabolism research shows that exercise elicits broad metabolomic changes (notably in lipid pathways) and affects amino acid catabolism; specific metabolites (e.g., lactate and succinate) can regulate immune cell function. However, the long-term impact of exercise on circulating metabolites and immunity is not well defined. The authors hypothesize that early-life exercise induces lasting epigenetic and metabolic reprogramming—particularly in hepatic lysine degradation and pipecolic acid production—that enhances anti-inflammatory immunity in midlife.

• Epidemiological and clinical literature links regular physical activity with reduced infection incidence and severity, including COVID-19, and mitigated chronic inflammation.
• Longitudinal data (Amsterdam Growth and Health Longitudinal Study) and retrospective studies indicate that youth physical activity predicts favorable adult health metrics (fitness, bone mineral density, adiposity, cardiometabolic disease risk).
• Metabolomics studies show acute exercise provokes robust changes across lipid super-pathways and branched-chain amino acid catabolism, with downstream effects on insulin sensitivity and metabolic risk.
• Immunometabolic intermediates influence immunity: lactate can induce pro-inflammatory T-cell phenotypes and anti-inflammatory monocyte phenotypes; succinate triggers inflammatory responses. While acute exercise alters these intermediates, long-term exercise effects on metabolome-immune interactions were previously unclear.
• Animal studies demonstrate pre-exercise mitigates LPS-induced lung injury; yet durable, early-life exercise effects on later-life immunity and the molecular mediators had not been fully explored.

• Animals: Male C57BL/6J mice began at 1 month of age.
• Exercise protocol: Swimming 60 min/day, 5 days/week for 12 weeks (with 1-week adaptation), then detraining (no training) until 15 months of age. Age-matched sedentary controls underwent brief daily swim (30 s) and drying.
• Sepsis model: Single intraperitoneal injection of ultrapure E. coli LPS (2 mg/kg). Assessed at 0, 2, 4, 6, 12, 24, 48, 72 h.
• Clinical and physiological measures: Murine sepsis score (MSS), body weight, body temperature, blood glucose.
• Hematology and cytokines: Complete blood counts (WBCs, granulocytes, lymphocytes, monocytes). Serum cytokines assessed by array and ELISA (TNF, IL-1β, IL-1Ra; and others in supplementary data).
• Histology and immunostaining: H&E and Oil Red O staining of liver; lung histology; F4/80 immunofluorescence for hepatic macrophage infiltration.
• Metabolomics: Untargeted LC-MS metabolomics (serum at 4 and 15 months; liver at 15 months). Multivariate analysis with OPLS-DA; significance criteria p<0.05 and VIP≥1. Targeted LC-MS/MS quantified pipecolic acid in serum and liver.
• Human studies: (1) 27 young healthy volunteers (22.85 ± 3.40 years) underwent a single-bout submaximal cycling exercise; serum pipecolic acid measured pre/post. (2) Cross-sectional comparison of serum pipecolic acid in 18 young highly trained athletes (team-based skiing >1 year) vs 21 inactive healthy individuals (all male, 16.21 ± 2.80 years).
• Pipecolic acid intervention in mice: Intraperitoneal pipecolic acid (200 µg/kg) administered 1 h before and 24 h after LPS; targeted LC-MS confirmed elevation.
• Macrophage experiments: Bone marrow-derived macrophages (BMDMs) treated with LPS (100 ng/ml, 6 h) ± pipecolic acid (5, 10, 20 µM). qRT-PCR for TNF, IL-1β, iNOS. RNA-seq with KEGG and GSEA analyses. Western blot for mTORC1 signaling (p-p70S6K Thr389, p-S6 Ser240/244, p-mTOR Ser2448). Rapamycin (100 nM) co-treatment to probe pathway involvement.
• Genetic manipulation in vivo: Liver-specific knockdown of Crym via AAV8-shRNA or scramble control injected at 14 months in previously exercised or sedentary mice; LPS challenge at 15 months. Verified Crym protein knockdown; measured serum and liver pipecolic acid; assessed clinical, hematologic, cytokine and histologic endpoints.
• Epigenetic assays: Global DNA methylation and targeted analysis of Crym promoter CpG island (no change). Histone marks (H3K4me3, H3K27me3, H3K27ac) by Western blot in liver. ChIP-seq for H3K4me3 occupancy at Crym promoter; ChIP-qPCR in primary hepatocytes (multiple promoter regions, notably −366 to −190 bp).
• Hepatocyte mechanistic studies: Primary mouse hepatocytes transfected with Ad-SETD1A or Ad-eGFP, or si-SETD1A vs si-Ctrl; assessed SETD1A levels, H3K4me3 enrichment at Crym promoter, and Crym mRNA/protein expression.
• Statistics: Normality by Shapiro–Wilk; two-tailed Student’s t-test or Mann–Whitney for two-group comparisons; one-way or two-way (repeated measures) ANOVA with Bonferroni post hoc tests; p<0.05 significant. Typical n=6–8 per group for animal endpoints; details provided per figure.
• Ethics: Human and animal protocols approved by relevant ethics committees; informed consent obtained.

• Early-life exercise confers durable protection against endotoxemia: Mice trained by swimming from 1–4 months exhibited, at 15 months after 11 months detraining, significantly lower sepsis severity (reduced MSS at 12, 24, 48 h post-LPS), and faster recovery of body weight, body temperature, and blood glucose after LPS (2 mg/kg i.p.; n=8/group; p<0.05).
• Hematologic and inflammatory profiles: Exercised mice showed higher WBC, lymphocyte, and monocyte counts at 6 h post-LPS and reduced systemic inflammation at 48 h (lower serum TNF and IL-1β; higher IL-1Ra). Liver and lung histology showed reduced inflammatory infiltration and hepatic lipid accumulation.
• Metabolomics highlighted pipecolic acid: Untargeted LC-MS with OPLS-DA revealed distinct serum profiles at 4 and 15 months; pipecolic acid was the only metabolite consistently upregulated in exercised mice at both ages. Liver metabolomics at 15 months also ranked pipecolic acid among top differentials (VIP≥1, p<0.05). Targeted LC-MS/MS confirmed higher pipecolic acid in serum and liver at both time points.
• Human relevance: Serum pipecolic acid increased after a single bout of submaximal exercise in healthy young volunteers (n=27). Young highly trained athletes (n=18) had higher serum pipecolic acid than untrained peers (n=21).
• Pipecolic acid therapy recapitulates benefits: In mice, pipecolic acid (200 µg/kg i.p., −1 h and +24 h relative to LPS) elevated circulating pipecolic acid, increased WBCs at 6 h, reduced MSS (12–48 h), accelerated recovery of glucose, body weight, and temperature, lowered serum TNF and raised IL-1Ra at 24 h, and reduced hepatic and pulmonary inflammatory injury (n≈6–8/group; p<0.05).
• Macrophage mechanism via mTORC1: In LPS-stimulated BMDMs, pipecolic acid dose-dependently reduced TNF, IL-1β, and iNOS mRNAs (5–20 µM). RNA-seq/KEGG/GSEA indicated mTORC1 pathway inhibition; Western blots confirmed reduced p-p70S6K (Thr389) and p-S6 (Ser240/244). Rapamycin co-treatment did not further suppress cytokines, supporting mTORC1 involvement.
• Hepatic Crym is required: Among lysine/ pipecolic acid pathway genes, Crym was selectively upregulated (mRNA and protein) in livers of exercised mice. Liver-specific Crym knockdown (AAV8-shRNA) in exercised mice abolished the exercise-induced elevation of serum and liver pipecolic acid and eliminated protection against LPS (no differences vs sedentary with Crym knockdown across WBC responses, recovery metrics, cytokines, and histology).
• Epigenetic basis: Early-life exercise increased H3K4me3 in liver globally and at the Crym promoter (ChIP-seq and ChIP-qPCR, notably −366 to −190 bp). DNA methylation of the Crym promoter was unchanged. SETD1A expression was elevated in hepatocytes from exercised mice (at 4 and 15 months). SETD1A overexpression increased H3K4me3 at the Crym promoter and increased Crym expression; SETD1A knockdown showed the opposite, indicating SETD1A-driven H3K4me3 promotes Crym and pipecolic acid production.

The findings demonstrate that a finite period of early-life exercise establishes a long-lasting, beneficial immunometabolic state that improves resilience to systemic inflammatory challenge in midlife. The work links exercise to durable epigenetic remodeling in the liver—specifically increased H3K4me3 at the Crym promoter via SETD1A—which elevates hepatic production of pipecolic acid, a lysine-derived metabolite. Pipecolic acid, in turn, exerts anti-inflammatory effects by suppressing mTORC1 signaling in macrophages, reducing pro-inflammatory cytokine expression, and improving clinical outcomes in LPS-induced sepsis. These results address a key gap concerning how exercise history can condition long-term immune function and reveal pipecolic acid as a mediator connecting exercise, metabolism, and innate immune regulation. Human observations (acute exercise and trained athletes) corroborate that exercise elevates circulating pipecolic acid, suggesting translational relevance. The study situates exercise-induced immunometabolic programming within broader concepts of trained immunity and highlights mTORC1 as an effector pathway modulated by a specific exercise-responsive metabolite.

Early-life regular exercise enhances anti-inflammatory immunity well into middle age in mice through epigenetic immunometabolic reprogramming. The mechanism centers on increased hepatic pipecolic acid production driven by SETD1A-mediated H3K4me3 enrichment at the Crym promoter, leading to suppression of macrophage mTORC1 signaling and dampened cytokine responses to LPS. Pipecolic acid supplementation mimics these benefits. Future research should: (1) evaluate whether early-life exercise confers long-term immunoprotection in humans and whether pipecolic acid tracks with these benefits during detraining and aging; (2) test live-pathogen and polymicrobial sepsis models to generalize findings; (3) dissect additional pathways (e.g., TNF signaling) influenced by pipecolic acid; and (4) establish causal in vivo links between H3K4me3 remodeling and Crym expression in liver across species.

• Species and model limitations: Long-term effects were demonstrated in mice; human data are limited to acute and cross-sectional associations of pipecolic acid with exercise, not direct long-term immune outcomes.
• Sepsis model: Endotoxemia (LPS) was used rather than live pathogen or polymicrobial models, limiting assessment of host–pathogen interactions.
• Epigenetic causality: While SETD1A-mediated H3K4me3 enrichment at the Crym promoter associates with increased Crym expression, in vivo causal verification and broader epigenomic mechanisms require further study.