Researchers are exploring whether these ubiquitous fluorinated molecules might worsen infections or hamper vaccine effectiveness.
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Edited by Frederick M. Ausubel, Harvard Medical School and Massachusetts General Hospital, Boston, MA, and approved July 21, 2017 (received for review April 19, 2017)
Article Figures & SI
Figures
- Fig. 1.
Commensal E. coli-derived indole extends lifespan and healthspan in C. elegans and Drosophila melanogaster. (A) Kaplan–Meier lifespan curves of wild-type C. elegans N2 on E. coli K12, which produces indole, or E. coli K12ΔtnaA, which does not. Graph represents seven independent experiments, n > 500 worms per condition). (B) Maximal lifespan obtained from at least three independent experiments comparing K12 vs. K12ΔtnaA or K12ΔtnaA plus indole (Indole) vs. K12ΔtnaA plus vehicle (Vehicle). (C) Lifespan curves of N2 on K12ΔtnaA supplemented with either 250 μM indole or vehicle (Methanol). Graph combines three independent experiments with n > 490 worms per condition. (D) Kaplan–Meier lifespan curves of N2 on K12 supplemented with either 100 μM indole or vehicle (n > 150 animals per condition). (E) Survival curves of N2 at 35 °C (day 2 or day 18) from K12ΔtnaA plus vehicle (Vehicle) or K12ΔtnaA plus indole (Indole). n = 25 animals per condition. (F) Survival of day 4 adult N2 animals at 35 °C grown in vehicle or indole without bacteria. (G) Thrashing motility in liquid in N2 adults grown on either K12ΔtnaA plus vehicle (Vehicle) or K12ΔtnaA plus indole (Indole), and monitored throughout life (n = 8–12 worms); (H) pharyngeal pumping rate per minute in N2 animals grown as in G (n = 8–12 worms); (I) paralysis events in N2 adults grown as in G and H (n = 200 worms per condition). (J) Healthspan and frail span measurements of N2 adults grown either on K12ΔtnaA-vehicle (Vehicle) or K12ΔtnaA-indole (Indole) conditions. Values for motility in liquid, pharyngeal pumping rate, and paralysis events were obtained from G, H, and I, respectively. (K) Healthspan values normalized with maximum lifespan set at 100%. (L) Lifespan of germ-free wild-type Drosophila melanogaster w1118 monoassociated with either K12 or K12ΔtnaA (n > 60 animals per condition). (M) Climbing assay of young (2 d), and older (20 d) adult germ-free w1118 flies monoassociated with either K12 or K12ΔtnaA (n > 30 animals per condition). (N) Heat stress resistance assay with conventionally raised streptomycin-treated w1118 flies, fed either K12, or K12ΔtnaA, or vehicle (Methanol) or 250 μM indole (n > 30 animals per condition). G–I and L–N are representative of at least two independent experiments. P values of percent survival curves were calculated with log-rank test. Values of B, F–I, and M and N represent mean values ± SEM, and t tests were performed to calculate P values. *P < 0.05; **P < 0.01; ***P < 0.001. Summary of the lifespan experiments are presented in Table S3.
- Fig. S1.
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).
- Fig. 2.
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.
- Fig. S2.
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.
- Fig. 3.
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.
- Fig. S3.
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.
- Fig. S4.
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.)
- Fig. 4.
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.
- Fig. 5.
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.
- Fig. S5.
Mouse motility in geriatric BALB/c animals colonized with K12 or K12ΔtnaA. Motility tracking of geriatric animals for 2 min. The y axis represents the long side of a cage in centimeters, and the x axis represents the short side of the cage in centimeters. The images show representative tracks from two geriatric animals one colonized with K12 (Left), and the other with K12ΔtnaA (Right) for 90 d. From these data, the total distance traveled was calculated.
Tables
Figures Conditions Maximum lifespan + SEM, d Median lifespan + SEM, d N Censored P value Fig. 1A N2 + K12ΔtnaA 25.5 ± 1.0 17.2 ± 0.8 534 15 <0.0001 N2 + K12 27.7 ± 1.1 20.2 ± 0.7 677 15 Fig. 1C N2 + K12ΔtnaA + vehicle 29.0 ± 1.7 18.2 ± 0.5 542 3 <0.0001 N2 + K12ΔtnaA + indole 29.6 ± 1.2 22.4 ± 1.2 492 5 Fig. 1D N2 + K12 + vehicle 32.5 ± 0.5 22 ± 1 226 0 <0.0001 N2 + K12 + indole 36.5 ± 1.5 25.5 ± 1.5 243 0 Fig. 1L W1118 + K12ΔtnaA 103.0 ± 2.0 90.5 ± 2.5 116 0 <0.0001 W1118 + K12 110.0 ± 4.0 100.0 ± 5.0 60 0 Fig. 2A ahr-1(ju145) + K12ΔtnaA 31.3 ± 0.6 22.8 ± 1.5 117 12 0.15 ahr-1(ju145) + K12 32.0 ± 0.6 22.3 ± 1.2 112 5 Fig. 2B ahr-1(ia3) + K12ΔtnaA 33.0 ± 0.7 24.8 ± 0.8 95 0 0.065 ahr-1(ia3) + K12 31.5 ± 0.5 23.0 ± 0.9 104 0 Fig. 2D ahr-1(ju145) + K12ΔtnaA + vehicle 28.7 ± 2.0 24.0 ± 2.0 98 1 0.051 ahr-1(ju145) + K12ΔtnaA + indole 29.0 ± 1.7 22.7 ± 2.3 111 1 Fig. 2E ahr-1(ia3) + K12ΔtnaA + vehicle 30.5 ± 1.5 21.5 ± 1.5 368 3 0.36 ahr-1(ia3) + K12ΔtnaA + indole 30.5 ± 1.5 20 ± 2 258 2 Fig. S2B sir-2.1(ok434) + K12ΔtnaA 22.0 ± 0.0 18.0 ± 0.0 79 0 <0.0001 sir-2.1(ok434) + K12 24.0 ± 0.0 21.0 ± 1.0 93 0 Fig. S2C skn-1(ju169) + K12ΔtnaA 19.0 ± 0.0 12.5 ± 0.5 80 0 <0.0001 skn-1(ju169) + K12 19.0 ± 0.0 15.0 ± 1.0 79 0 Fig. S2D daf-16(m26) + K12ΔtnaA 23.0 ± 0.0 18.0 ± 0.0 88 7 <0.0001 daf-16(m26) + K12 23.0 ± 0.0 20.3 ± 0.3 59 5 Fig. S2E cat-2(e1112) + K12ΔtnaA 21.3 ± 0.7 17.6 ± 1.55 71 0 <0.0001 cat-2(e1112) + K12 24.0 ± 1.0 20.0 ± 0.0 56 2 Fig. S2F dop-3(vs.106) + K12ΔtnaA 30.0 ± 0.0 22.0 ± 1.0 91 0 <0.0001 dop-3(vs.106) + K12 30.0 ± 0.0 25.0 ± 0.0 90 0 P values were obtained from log-rank (Mantel–Cox) test.
- Table S1.
FPKM values of ahr-1 and fmo-2 in young and old N2 animals grown in K12 or K12ΔtnaA and FPKM values of fmo-2 in young and old ahr-1(ia3) animals grown in K12 or K12ΔtnaA
Gene Conditions FPKM values ahr-1 N2-young + K12ΔtnaA 7.1, 3.2 ahr-1 N2-young + K12 4.7, 14.3 ahr-1 N2-old + K12ΔtnaA 28.9, 33.9, 23.1 ahr-1 N2-old + K12 47, 31.3 fmo-2 N2-young + K12ΔtnaA 32.5, 25.8 fmo-2 N2-young + K12 55.65, 48.5 fmo-2 N2-old + K12ΔtnaA 505.8, 375.4, 518.5 fmo-2 N2-old + K12 413.8, 397.2 fmo-2 ahr-1(ia3)-young + K12ΔtnaA 83.5, 63.9, 74.5 fmo-2 ahr-1(ia3)-young + K12 54.9, 62.8, 83.1 fmo-2 ahr-1(ia3) + K12ΔtnaA 1,251.3, 681.5, 756.2 fmo-2 ahr-1(ia3)-old + K12 439.5, 951.5, 357.2 Sample ID Read length No. of reads % Total aligned % Unaligned % Stranded 14_N2tnaA_LATE2 1 × 151 17,077,925 98.53 1.47 99.57 15_N2tnaA_LATE3 1 × 151 11,568,564 91.67 8.33 99.57 16_N2K12_LATE1 1 × 151 17,635,082 98.41 1.59 99.67 18_N2K12_LATE3 1 × 151 16,765,410 97.66 2.34 99.66 2_N2tnaA_EARLY2 1 × 151 17,265,225 98.98 1.02 99.82 37_AHRK12_LATE1 1 × 151 13,349,441 96.24 3.76 99.58 39_AHRK12_LATE3 1 × 151 14,609,013 96.54 3.46 99.65 3_N2tnaA_EARLY3 1 × 151 16,956,473 98.84 1.16 99.81 4_N2K12_EARLY1 1 × 151 16,414,814 98.70 1.30 99.81 5_N2K12_EARLY2 1 × 151 16,813,260 98.88 1.12 99.76 6_N2K12_EARLY3 1 × 151 16,406,106 98.79 1.21 99.80
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Supporting Information
Indoles and healthspan
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