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Disease tolerance mediated by microbiome E. coli involves inflammasome and IGF-1 signaling

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Science  30 Oct 2015:
Vol. 350, Issue 6260, pp. 558-563
DOI: 10.1126/science.aac6468
  • Fig. 1 Effects of E. coli O21:H+ on DSS-induced skeletal muscle atrophy.

    (A and B) Jax mice were treated with 5% DSS. (A) Percent original weight. (B) Leg muscle mass at 7 days. Representative experiment (n = 4 to 6 independent experiments) using five animals per group, mean ± S.D. Unpaired t test. (C) Percent original weight of colony-born C57Bl/6 mice (University of California, Berkeley) treated with ampicillin, vancomycin, neomycin, metronidazole, and 5% DSS (methods). Representative experiment (n = 3 independent experiments) using four animals per group, mean ± SD. Unpaired t test. (D) Percent original weight of 5% DSS–treated Jax mice gavaged with 5 × 108 colony-forming units (CFU) E. coli O21:H+ or vehicle alone (methods). Representative experiment (n = 6 independent experiments) using four animals per group, mean ± SD. Unpaired t test. Untreated control mice ± E. coli O21:H+ are shown in fig. S3. (E and F) Germ-free Swiss Webster (SW) mice gavaged with 5 × 108 E. coli O21:H+ or E. coli MG1655 (supplementary materials, materials and methods) and treated with 5% DSS. (E) Percent original weight. (F) Leg muscle mass at 5 days from a subset of mice in (E) that were weight matched at day 0. Representative experiment (n = 4 independent experiments) using five to seven animals per group, mean ± SD. Unpaired t test. Monocolonized SW controls are shown in fig. S3. (G) Percent survival of Jax mice administered E. coli O21:H+ or heat-killed E. coli O21:H+ (supplementary materials, materials and methods) and treated with 5% DSS for 7 days, after which DSS was no longer administered. Results are from two independent experiments using four to seven mice per group per experiment. Log rank analysis. Survival analyses of untreated control ± E. coli O21:H+ mice have been carried out for 1 year with no deaths from either group. Vehicle/DSS–treated mice exhibit similar death kinetics as heat-killed/DSS treated mice. ****P < 0.0001, ***P < 0.0005, **P < 0.005, *P < 0.05. EDL, extensor digitorum longus; TA, tibialis anterior. Experiments terminated for postmortem analyses at indicated day when one or more mice lost [(A), (B), (D), (E), and (F)] >15% body weight in accordance with our Salk animal protocol or (C) >20% weight loss in accordance with our animal protocol (University of California, Berkeley).

  • Fig. 2 Effects of E. coli O21:H+ colonization on muscle wasting induced by infections.

    (A) Percent original weight of Jax mice orally infected with 8.5 × 106 S. Typhimurium. Representative experiment (n = 3 independent experiments) using five animals per group, mean ± SD. Unpaired t test. (B) Leg muscle mass from S. Typhimurium–infected mice in (A) at day 5 (ST) or B. thailandensis–infected mice in (E) at day 2 (Bt). Representative experiment (n = 3 independent experiments) using five animals per group, shown as mean ± SD. Unpaired t test. (C) Percent original weight of Jax mice gavaged with 5 × 108 CFU E. coli O21:H+ or vehicle alone and orally infected with 2 × 107 S. Typhimurium. Representative experiment (n = 7 independent experiments) using four to five animals per group, mean ± S.D. Unpaired t test. Untreated control mice ± E. coli O21:H+ are shown in fig. S3. (D) Leg muscle mass from infected E. coli O21:H+ or vehicle-control mice from (C) at day 3 (ST) and from (F) at day 2 (Bt). Representative experiment (n = 3 to 7 independent experiments) using four to five animals per group, mean ± SD. Unpaired t test. Control mice ± E. coli O21:H+ are shown in fig. S3. (E and F) Jax mice were (E) left untreated or (F) gavaged with 5 × 108 CFU E. coli O21:H+ or vehicle alone (supplementary materials, materials and methods) and intranasally infected with 2500 CFU B. thailandensis. Representative experiment (n = 3 to 7 independent experiments) using four to five animals per group, shown as mean ± SD. Unpaired t test. (G) S. Typhimurium (2 × 107 CFU inoculum) or B. thailandensis (2500 CFU inoculum) burdens in target tissues from infected mice ± E. coli O21:H+ at 3 days (ST) or 2 days (Bt). B. thailandensis was not detected in the liver and spleen at this dose. Representative experiment (n = 3 to 7 independent experiments) using three to seven animals per group, mean ± SD. Unpaired t test. Dotted line indicates limit of detection. (H) Percent survival of Jax mice treated with 5 × 108 CFU E. coli O21:H+ or vehicle-control orally infected with 2 × 107 S. Typhimurium. Results are from two independent experiments with 10 to 12 animals per group per experiment. Log rank analysis. ****P < 0.0001 ***P < 0.0005, **P < 0.005, *P < 0.05. Postmortem analyses at day when one or more mice lost >15% body weight. EDL, extensor digitorum longus; TA, tibialis anterior.

  • Fig. 3 Gut colonization by E. coli O21:H+ associated with down-regulation of muscle atrophy programs and maintenance of IGF-1 signaling during infection.

    (A and B) Jax mice gavaged with 5 × 108 CFU E. coli O21:H+ or vehicle alone and intranasally infected with 1000 CFU B. thailandensis. (A) Murf1 and Atrogin-1 expression in leg muscle at 2 days after infection normalized to ribosomal protein S17 (RPS17) expression. Representative experiment (n = 3 independent experiments) using four to nine animals per group, mean ± SD [analysis of variance (ANOVA)]. (B) Serum IGF-1 at 2 days. Representative experiment (n = 3 independent experiments) using seven to nine animals per group, shown as mean ± SD (ANOVA). Additional controls are provided in fig. S14. (C and D) Jax mice were given 5 × 108 CFU E. coli O21:H+ or vehicle alone and intranasally infected with 2500 CFU B. thailandensis. (C) IGF-1 levels in WAT and (D) E. coli O21:H+ levels in the WAT at day 2. Representative experiment (n = 4 independent experiments) using six to ten animals per group, mean ± SD (ANOVA). Red line indicates the limit of detection. (E to H) Mice given 5 × 108 CFU E. coli O21:H+ or vehicle alone were injected intraperitoneally with antibody to IGF-1 or IgG isotype control antibody and intranasally infected with 1000 CFU B. thailandensis. (E) Percent original weight. Results are from two independent experiments using six to nine mice per group per experiment (12 to 17 mice total), mean ± SD (ANOVA). Additional controls are provided in fig. S14. (F) Leg muscle mass and (G) adipose tissue mass at 2 days. EDL, extensor digitorum longus; TA, tibialis anterior; GWAT, gonadal white adipose tissue; RPWAT, retroperitoneal WAT; IWAT, inguinal WAT; BAT, brown adipose tissue. Results are from the two independent experiments described in (E) using five to nine mice weight-matched at day 0 (~18 g) for each group for each experiment (10 to 14 mice total), mean ± SD (ANOVA). (H) Murf1 and Atrogin-1 expression in leg muscle at 2 days and normalized to RPS17. Representative experiment (n = 2 independent experiments) using five to nine animals per group, mean ± SD (ANOVA). ****P < 0.0001, ***P = 0.0006, **P < 0.005, *P < 0.05. Postmortem analyses were performed at day when one or more mice lost >15% body weight.

  • Fig. 4 The Nlrc4 inflammasome is required for sustaining IGF-1 levels and mediates the E. coli O21:H+–associated protection against muscle wasting promoted by pathogenic infection.

    (A to C) Wild-type and Casp1−/−11−/− mice were administered 5 × 108 CFU E. coli O21:H+ or vehicle alone and then intranasally infected with 2500 CFU B. thailandensis as described in the supplementary materials, materials and methods. (A) Percent original weight at day 2. Additional controls are provided in fig. S14. (B) Expression of Murf1 and Atrogin-1 was determined in leg muscle at 2 days after infection normalized to expression levels of RPS17. (C) Serum IGF-1 quantified at day 2. Representative experiment (n = 3 independent experiments) using 10 animals per wild-type group and five to seven mice per Casp1−/−11−/− group, mean ± S.D. (ANOVA). (D) Wild-type and Nlrc4−/− mice were given 5 × 108 CFU E. coli O21:H+ or vehicle alone and infected intranasally with 2500 CFU B. thailandensis. Serum levels of IGF-1 at 2 days after infection were measured. Representative experiment (n = 2 independent experiments) using 10 animals per wild-type group and five mice per Nlrc4−/− group, shown as mean ± SD (ANOVA). (E) Jax mice were given 5 × 108 CFU E. coli O21:H+ or vehicle alone and then intranasally infected with 2500 CFU B. thailandensis as described in the supplementary materials, materials and methods, and serum IL-18 levels were measured at 2 days after infection. Representative experiment (n = 2 independent experiments) using 10 animals per group, mean ± SD (ANOVA). (F) Wild-type and Il1β−/−Il18−/− mice were administered 5 × 108 CFU E. coli O21:H+ or vehicle alone and then intranasally infected with 2500 CFU B. thailandensis. Serum levels of IGF-1 at 2 days after infection were measured. Representative experiment (n = 3 independent experiments) using 10 animals per wild-type group and eight mice per Il1β−/−Il18−/− group, mean ± SD (ANOVA). ***P < 0.0003, **P < 0.006, *P < 0.05. Experiment was terminated at 2 days for postmortem analyses when one or more mice lost >15% body weight in accordance with our Salk animal protocol. Additional controls for (C), (D), and (F) are provided in fig. S14.

Supplementary Materials

  • Disease tolerance mediated by microbiome E. coli involves inflammasome and IGF-1 signaling

    Alexandria M. Palaferri Schieber, Yujung Michelle Lee, Max W. Chang, Mathias Leblanc, Brett Collins, Michael Downes, Ronald M. Evans, Janelle S. Ayres

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S19
    • Table S1
    • Full Reference List
    Table S2

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