Citrulline regulates macrophage metabolism and inflammation to counter aging in mice

Abstract

Metabolic dysregulation and altered metabolite concentrations are widely recognized as key characteristics of aging. Comprehensive exploration of endogenous metabolites that drive aging remains insufficient. Here, we conducted an untargeted metabolomics analysis of aging mice, revealing citrulline as a consistently down-regulated metabolite associated with aging. Systematic investigations demonstrated that citrulline exhibited antiaging effects by reducing cellular senescence, protecting against DNA damage, preventing cell cycle arrest, modulating macrophage metabolism, and mitigating inflammaging. Long-term citrulline supplementation in aged mice yielded beneficial effects and ameliorated age-associated phenotypes. We further elucidated that citrulline acts as an endogenous metabolite antagonist to inflammation, suppressing proinflammatory responses in macrophages. Mechanistically, citrulline served as a potential inhibitor of mammalian target of rapamycin (mTOR) activation in macrophage and regulated the mTOR-hypoxia-inducible factor 1α-glycolysis signaling pathway to counter inflammation and aging. These findings underscore the significance of citrulline deficiency as a driver of aging, highlighting citrulline supplementation as a promising therapeutic intervention to counteract aging-related changes.

Fig. 1.. Citrulline is significantly down-regulated during aging.

(A) Graphic illustration of the workflow to profile age-associated metabolites in brain tissue, liver tissue and serum from mice at different ages using LC-MS. Age-associated metabolites were screened by Kruskal-Wallis test (P < 0.05) and Spearman correlation (P < 0.05). w, weeks; m/z, mass/charge ratio. (B) Pathway enrichment analyses of age-associated metabolites from mouse brain, liver, and serum (hypergeometric test, ***P < 0.001, **P < 0.01, and *P < 0.05). TCA, tricarboxylic acid. (C) Age-associated metabolites were screened by Kruskal-Wallis test (P < 0.05) and Spearman correlation (P < 0.05). Red dots (brain, n = 26; liver, n = 46; serum, n = 21) and blue dots (brain, n = 49; liver, n = 60; serum, n = 59) represent metabolites that were increased and decreased over the age, respectively. Black dots represent unchanged metabolites. (D) Venn diagram for the overlap of age-associated metabolites in mouse brain, liver, and serum obtained from (C). (E) The spearman factors of seven common age-associated metabolites in mouse brain, liver, and serum datasets. Red and blue bars represent metabolites that were increased and decreased over the age, respectively. n = 3. Three dots for each metabolite represent their Spearman factors with age obtained from mouse brain, liver, and serum datasets, respectively. (F) Relative abundances of citrulline in mouse brain, liver, and serum at different ages. Bars represent means ± SEM. n = 8 to 10; all numbers are biologically independent samples. ***P < 0.001, **P < 0.01, and *P < 0.05; n.s., not significant (two-tailed Student’s t test).

Fig. 2.. Citrulline supplement mitigates age-associated phenotypes in vitro and ex vivo.

(A) Schematic illustration of senescent immortalized MEF (iMEF) generation using tBHP. n = 6 to 7. h, hours; d, days. (B) Representative images illustrating γH2AX (green) levels responding to citrulline (Cit; 500 μM for 4 days) in iMEFs with 4′,6-diamidino-2-phenylindole (DAPI)–stained nuclei (blue). n = 3; each point averaged from 3 images. Scale bar, 50 μm. (C) Representative SA-β-Gal activity staining images in iMEFs responding to citrulline (500 μM for 4 days). Scale bar, 50 μm. (D) p21 mRNA levels in iMEFs responding to citrulline (500 μM for 4 days). n = 6. (E) mRNA levels of Tnf, Il6, and Il1b in iMEFs responding to citrulline (500 μM for 4 days). n = 5 to 6. (F) Graphical illustration of BMDM generation and citrulline levels in BMDMs. n = 5. (G) Representative images illustrating γH2AX (green) levels responding to citrulline (500 μM for 24 hours) in BMDMs with DAPI-stained nuclei (blue). n = 3; each point averaged from three images. Scale bar, 100 μm. (H) Representative SA-β-Gal activity images in aged BMDM cells after citrulline treatment (500 μM for 24 hours). n = 3. Scale bar, 10 mm. (I) mRNA levels of Tnf, Il6, and Il1b in BMDMs after citrulline treatment (500 μM for 24 hours). n = 3 to 4. (J) mRNA levels of TNF and IL1B in young (19 years old, male) and aged (56 years old, male) human macrophages after citrulline treatment (500 μM for 12 hours). n = 6 to 8. Bars represent means ± SEM. ***P < 0.001, **P < 0.01, and *P < 0.05 (two-tailed Student’s t test).

Fig. 3.. Beneficial effects of long-term citrulline supplementation in aging mice.

(A) The long-term supplementation scheme of young and aged mice (male) with drinking water only or drinking waters containing citrulline (1 g/kg of body weight of mice) for about 9 weeks (6 and 72 weeks old, male). n = 8. (B) Recorded body weights of young and aged mice (male) supplemented with water or citrulline over 9 weeks. n = 8. (C) Weights of spleen and liver organs in young and aged mice (male) supplemented with water or citrulline over 9 weeks. n = 6 to 8. (D to F) Real-time polymerase chain reaction (PCR) analyses of senescence-associated secretory phenotype genes, including Tnf, Il6, and Il1b in mouse brain (D), liver (E) and primary BMDMs (F). n = 5 to 6. (G and H) Representative immunofluorescence images of γH2AX (G) and P21 (H) levels in brain tissues of young and aged mice (male) supplemented with water or citrulline over 9 weeks. The microglia in the brain were stained with anti-IBA1 antibody (green), and the nuclei were stained with DAPI (blue). Scale bars, 50 μm. (I and J) The averaged density of positive cells in images from (G) and (H). n = 3; each point represents the mean densities of three images in each sample. Bars represent means ± SEM. ***P < 0.001, **P < 0.01, and *P < 0.05 (two-tailed Student’s t test).

Fig. 4.. Citrulline is an endogenous metabolite antagonist to inflammation.

(A) Tnf, Il6, and Il1b levels in iBMDMs responding to citrulline (4.5 hours) and LPS (100 ng/ml for 4 hours). n = 12, three samples with four technical replicates. (B) TNFα and IL-6 levels in iBMDM culture medium responding to citrulline (4.5 hours) and LPS (100 ng/ml for 4 hours). n = 5 to 8. (C) The scheme of mice administrated for 7 days. (D and E) Mouse survival after LPS challenge using log-rank (Mantel-Cox) test. (F) Citrulline levels in mice treated with citrulline and LPS (20 mg/kg). n = 8. (G) Tnf, Il6, and Il1b levels in mouse liver treated with citrulline and LPS (20 mg/kg). n = 5 to 8. (H) TNFα, IL-6, and IL-1β levels in mouse serum treated with citrulline and LPS (20 mg/kg). n = 8 to 16. (I) Graphical illustration of BMDM generation and citrulline levels in BMDMs treated with LPS (100 ng/ml for 12 hours). n = 5 or 8. (J) Tnf, Il6, and Il1b levels in BMDMs responding to citrulline (500 μM for 12.5 hours) and LPS (100 ng/ml for 12 hours). n = 3 to 4. (K) Quantifications of SA-β-Gal activities in BMDMs responding to citrulline (500 μM for 12.5 hours) and/or LPS (100 ng/ml for 12 hours). n = 3. (L) Graphical illustration of human macrophages from PBMCs. (M) TNF, IL1B, and IL6 levels responding to citrulline (500 μM for 12.5 hours) and LPS (100 ng/ml for 12 hours) in human macrophages. n = 6 to 8. Bars represent means ± SEM. ***P < 0.001, **P < 0.01, and *P < 0.05 (two-tailed Student’s t test).

Fig. 5.. Citrulline modulates macrophage metabolism to counter aging and inflammation.

(A) BMDMs from mice were treated with citrulline (500 μM for 12 hours) for untargeted metabolomics. (B) Sixteen enriched metabolic pathways in aged BMDMs treated with citrulline (500 μM for 12 hours). CoA, coenzyme A. (C) Glycolysis gene levels in BMDMs treated with citrulline (500 μM for 12 hours). n = 3 to 4. (D) HIF1α expressions in BMDMs treated with citrulline (500 μM for 12 hours). n = 3 to 4. (E) p-mTOR, mTOR, p-p70S6k, p70S6k, p-4EBP1, and 4EBP1 expressions in BMDMs responding to citrulline (500 μM for 12 hours). (F) A proposed model for citrulline to counter aging and inflammation through modulating mTOR-HIF1α-glycolysis. (G) p-mTOR, mTOR, p-p70S6k, p70S6k, p-4EBP1, and 4EBP1 expressions r4esponding to citrulline (500 μM for 4.5 hours) and LPS (100 ng/ml for 4 hours) in iBMDMs. (H) HIF1α expressions in iBMDMs treated with citrulline (500 μM for 4.5 hours) and LPS (100 ng/ml for 4 hours). n = 3. (I) Glycolysis genes in iBMDMs treated with citrulline (500 μM for 4.5 hours) and LPS (100 ng/ml for 4 hours). n = 3. (J) ECAR of iBMDMs treated with citrulline (500 μM for 4.5 hours) and LPS (100 ng/ml for 4 hours). n = 6 to 11. (K and L) Representative images of p-S6 (K) and HIF1α (L) in mouse brain. Microglia were stained with anti-IBA1 antibody (green) and DAPI (blue). Scale bars, 50 μm. (M and N) Averaged density of positive cells in (K) and (L). n = 3; each point represents the mean from three images. Bars represent means ± SEM. ***P < 0.001, **P < 0.01, and *P < 0.05 (two-tailed Student’s t test).

Fig. 6.. Age-dependent down-regulation of Nos2 contributed to citrulline deficiency in macrophages.

(A) Schematic illustration of stable-isotope tracing metabolomics using [U-¹³C]-arginine as a tracer. Young and aged BMDMs (male mice) were treated with 0.4 mM [U-¹³C]-arginine for 24 hours. OTC, ornithine transcarbamylase; ASL, argininosuccinate lyase. Black dots represent ¹²C and red dots represent ¹³C. (B to E) Relative abundances of metabolites in citrulline metabolism, including arginine (B), ornithine (C), arginosuccinate (D), and citrulline (E), in young and aged BMDMs (male mice) after stable-isotope tracing. n = 7 biologically independent samples in each group. n.d., not detected. (F) Real-time PCR analyses of Nos2 mRNA expressions in citrulline metabolism in young and aged BMDMs (male mice) after stable-isotope tracing. Bar graphs represent means ± SEM. n = 5. (G and H) Relative citrulline levels in the small intestine (G) and kidney (H) of young and aged mice (male) determined by LC-MS. n = 3 to 4. (I to K) Relative citrulline levels in plasma and PBMC samples from healthy humans: plasma (I), PBMC (J), and NOS2 expression from PBMCs (K) in young and aged human. The NOS2 expressions were derived from the PBMC transcriptome of healthy human donors using RNA sequencing (42). Bars represent means ± SEM. n = 7 to 9. Bars represent means ± SEM. ***P < 0.001, **P < 0.01, and *P < 0.05 (two-tailed Student’s t test).