Indoles from commensal bacteria extend healthspan

Effects of indoles on healthspan. (A) Lifespan curve of N2 with K12, K12ΔtnaA, or OP50 (n > 60 worms per condition; P < 0.0001 for K12ΔtnaA vs. other strains; differences between K12 and OP50 were not significant). (B) Thrashing motility in liquid (n = 8–12 worms per condition); (C) pharyngeal pumping rate per minute (day 12 adults, n = 8–12 worms per condition) of N2 adults grown on K12 or K12ΔtnaA. (D) Thrashing motility of N2 animals grown on K12 animals supplemented with 100 µM indole or vehicle (day 20 adults, n = 12 worms).

The AHR mediates effects of indoles on healthspan. (A and B) Kaplan–Meier lifespan curves of ahr-1(ju145) and ahr-1(ia3) on K12 and K12ΔtnaA, respectively. (C) Maximum lifespan obtained from the lifespan experiments comparing K12 vs. K12ΔtnaA with at least six biological replicates. (D and E) ahr-1(ju145) and ahr-1 (ia3) lifespan on K12ΔtnaA supplemented with 250 μM indole or vehicle (Methanol). Lifespan curves are representative of at least three independent experiments (n > 60 worms per condition in each experiment). (F) Heat stress assays of day 2 ahr-1(ia3) at 35 °C grown on K12ΔtnaA plus vehicle (Vehicle) or K12ΔtnaA plus indole (Indole) (n = 25 animals/condition). (G) Thrashing motility in liquid (n = 8–12 worms/condition); (H) pharyngeal pumping rate per minute (n = 8–12 worms per condition) of ahr-1(ia3) adults grown on K12ΔtnaA plus vehicle (Vehicle) or K12ΔtnaA plus indole (Indole), and monitored throughout their lifespan. (I and J) Healthspan and frail span measurements of ahr-1(ia3) adults grown on K12ΔtnaA plus vehicle (Vehicle) or K12ΔtnaA plus indole (Indole). Values for motility in liquid and pharyngeal pumping rate were obtained from G and H, respectively, to calculate healthspan, and then normalized to maximal lifespan in J. (K and L) Climbing assays (K) and heat stress assays (L) of day 20 germ-free w1118 and ss¹ Drosophila fed K12 or K12ΔtnaA (n > 30 animals per condition). P values of percent survival curves were calculated by a log-rank test. Values in C, G, H, K, and L represent mean ± SEM. Summary of the lifespan experiments are presented in Table S3.

Genes associated with lifespan and stress response regulation in C. elegans do not mediate healthspan effect of indole. (A–F) Kaplan–Meier lifespan curves of wild-type C. elegans, N2 (A), sir2.1(ok434) (B), skn-1(zu169) (C), daf-16(m26) (D), cat-2(e1112) (E), dop-3(vs.106) (F), daf-2(e1370) (G) on E. coli K12 or K12ΔtnaA mutant; daf-2(e1370) on K12ΔtnaA plus vehicle or K12ΔtnaA plus indole (H) (n > 50 worms/condition). Graphs are representatives of at least two independent experiments. Details of the lifespan experiments are listed in Table S3.

Indole limits age-associated changes in gene expression in C. elegans. (A) Venn diagram indicating the schema for identification of TnaA- and Ahr-dependent genes and TnaA-dependent and Ahr-independent genes. (B) Hierarchical clustering of 494 z-score–normalized TnaA- and Ahr-dependent genes in young and old animals grown in K12 or K12ΔtnaA. Replicates in each condition are demarcated by dotted black line, while each of the conditions is demarcated by solid black line. (C) PCA using FPKM values of 494 TnaA- and Ahr-dependent genes in young and old animals grown in K12 or K12ΔtnaA. (D) Hierarchical clustering of 1,254 aging genes (aging and lifespan-associated genes from ref. 25 in young and old animals grown in K12 or K12ΔtnaA). (E) PCA using FPKM values of 1,254 aging genes in young and old animals grown in K12 or K12ΔtnaA.

Expression of ahr-1 and fmo-2 increases with age but not with indole. (A and B) Real-time PCR analysis of expression levels of ahr-1 (A) and fmo-2 (B) in young and old N2 animals grown in K12 or K12ΔtnaA. (C) fmo-2 expression level in young and old ahr-1(ia3) animals grown in K12 or K12ΔtnaA. act-1 expression levels were used to normalize the ahr-1 and fmo-2 mRNA levels.

Characterization of transcriptional responses from C. elegans. (A) Schematic for the identification TnaA-dependent and Ahr-independent genes. (B) Expression profile of TnaA-dependent and Ahr-independent genes in young and old animals grown in K12 or K12ΔtnaA through hierarchical clustering of genes based on their corresponding z-score values. The replicates in each condition are separated by dotted black line; conditions are demarcated by solid black line. (C) PCA with FPKM values of TnaA-dependent and Ahr-independent genes in young and old animals grown in K12 or K12ΔtnaA. (D) GO output with TnaA-dependent and Ahr-independent genes performed using GOAmigo software. (E) Images of spermatheca of day 12 adult N2 hermaphrodites grown in K12 or K12ΔtnaA, after mating with males whose sperm had been labeled with MitoTracker. (Scale bar, 50 µm.)

Indole extends reproductive span of C. elegans. (A) GO analysis of 494 TnaA- and Ahr-dependent genes using GOAmigo software. (B) Average number of embryos and oocytes (unfertilized) produced per day by N2 adults grown on K12 or K12ΔtnaA over time (n = 12 adult worms per condition). (C) Average number of oocytes produced by day 10 N2 adults in K12ΔtnaA plus indole or K12ΔtnaA plus vehicle (n = 12 worms per condition). (D) Average number of larvae obtained from mating day 10 N2 hermaphrodites grown in K12 and K12ΔtnaA with young males. (E) Average number of oocytes produced by day 10 N2 and ahr-1(ia3) adults in K12 and K12ΔtnaA conditions (n = 12 worms per condition). B–E are representative of at least two independent experiments.

Indole from commensal E. coli augments lifespan and extends healthspan of mice. (A) Colony-forming units per gram of bacteria in feces of C57BL/6 mice colonized with streptomycin- and nalidixic acid-resistant K12 or K12ΔtnaA. Mice were colonized 1 wk before TBI (12 Gy), and bacterial counts were assessed 1 d before TBI on plates containing streptomycin and nalidixic acid. (B) Survival of C57BL/6 mice (n = 15) following colonization with either K12 or K12ΔtnaA, and exposure to TBI. (C) Survival of C57BL/6 mice (n = 20) following TBI. Mice were treated with either ICA (150 mg⋅kg⁻¹⋅d⁻¹) or vehicle, daily, beginning 1 d before irradiation. (D) Colony-forming units per gram of streptomycin/nalidixic acid-resistant K12 or K12ΔtnaA in feces of geriatric BALB/c mice (28 mo, n = 15). Bacterial counts were assessed at 30, 60, and 90 d postcolonization. (E) Urinary 3-indoxyl sulfate levels from geriatric mice colonized with either K12 or K12ΔtnaA. (F) Time course of weight changes in geriatric mice colonized with K12 or K12ΔtnaA. (G) Survival of geriatric mice colonized with K12 or K12ΔtnaA. (H) Composite health scores in geriatric mice colonized with K12 or K12ΔtnaA for 30, 60, or 90 d. (I) Average fraction of initial motility in geriatric BALB/c animals inoculated with K12 or K12ΔtnaA for 30, 60, or 90 d. Student’s t test only showed significant differences between groups at the 90-d time point.