Research ArticlePUBLIC HEALTH

A common antimicrobial additive increases colonic inflammation and colitis-associated colon tumorigenesis in mice

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Science Translational Medicine  30 May 2018:
Vol. 10, Issue 443, eaan4116
DOI: 10.1126/scitranslmed.aan4116
  • Fig. 1 Exposure to TCS increased basal inflammation in mice.

    Mice were treated with 10 or 80 ppm TCS in diet for 3 weeks. (A) Spleen weight (n = 7 per group). Ctrl, control. (B) Plasma concentration of IL-6 (n = 6 to 7 per group). (C) Gene expression of Il-6 in colon (n = 5 to 7 per group). (D) Left: Hematoxylin and eosin (H&E) staining of colon (magnification, ×40; n = 4 to 5 per group). Right: Histological score. The results are expressed as means ± SEM. *P < 0.05, **P < 0.01. The statistical significance was determined using one-way analysis of variance (ANOVA).

  • Fig. 2 Exposure to TCS increased DSS-induced colitis in mice.

    (A) Scheme of animal experiment. (B) Colon length (n = 6 to 12 per group). (C) Left: H&E staining of colon. Right: Histological score (magnification, ×40; n = 6 to 7 per group). (D) FACS quantification of immune cells in colon (see representative FACS plots in fig. S4A; n = 6 to 11 per group). (E) Concentration of IL-6 in plasma (n = 5 to 6 per group). (F) Gene expression of Il-6 in colon (n = 5 to 8 per group). (G) Effect of low-dose TCS (5 to 10 ppm in diet) on immune cell infiltration in colon (see representative FACS plots in fig. S4C; n = 6 to 8 per group). (H) Left: Effect of low-dose TCS on crypt damage in colon. Right: Histological score (magnification, ×40; n = 5 to 7 per group). The results are expressed as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. The statistical significance of two groups was determined using Student’s t test or Wilcoxon-Mann-Whitney test, and comparison of three groups was determined using one-way ANOVA.

  • Fig. 3 Exposure to TCS increased colitis in Il-10−/− mice.

    (A) Scheme of animal experiment. (B) FACS quantification of immune cells in colon (see representative FACS plots in fig. S5). (C) Gene expression of Il-6 in colon. (D) Left: H&E staining of colon (magnification, ×40). Right: Histological score. The results are expressed as means ± SEM. n = 5 to 6 per group. *P < 0.05. The statistical significance was determined using Student’s t test or Wilcoxon-Mann-Whitney test.

  • Fig. 4 Exposure to TCS increased AOM/DSS-induced colon tumorigenesis in mice.

    (A) Scheme of animal experiment. (B) Survival curve (n = 14 to 16 per group). (C) Quantification of colon tumor in mice (n = 5 to 7 per group). (D) Representative images of immunohistochemical staining of PCNA, β-catenin, and H&E and quantification of immunohistochemical images (n = 4 to 5 per group). (E) FACS quantification of immune cells in colon (see representative FACS plots in fig. S6B; n = 4 to 7 per group). (F) Concentrations of IL-6 and TNF-α in plasma (n = 5 to 6 per group). (G) Gene expression of Il-6 and Tnf-α in colon (n = 3 to 8 per group). (H) Effect of low-dose TCS (10 ppm in diet) on AOM/DSS-induced colon tumorigenesis in mice (n = 12 to 14 per group). (I) Effect of TCS (10 ppm in diet) on plasma concentration of TNF-α (n = 4 per group). (J) Effect of TCS (10 ppm in diet) on expression of Tnf-α in colon (n = 7 per group). The results are expressed as means ± SEM. *P < 0.05, **P < 0.01. The statistical significance was determined using Student’s t test or Wilcoxon-Mann-Whitney test, and the statistical analysis of survival was determined using log-rank (Mantel-Cox) test and Gehan-Breslow-Wilcoxon test.

  • Fig. 5 Exposure to TCS altered gut microbiota in mice.

    Mice were treated with 80 ppm TCS in diet for 3 weeks. (A) Effect of TCS on α diversity of fecal microbiota, assessed by PD whole tree analysis. (B) Effect of TCS on β diversity of fecal microbiota, assessed by principal coordinate (PC) analysis. (C) Effect of TCS on composition of the microbiota at phylum levels. (D) Effect of TCS on composition of the microbiota at genus levels. The results are expressed as means ± SEM. n = 12 to 15 per group. **P < 0.01. The statistical significance was determined using Student’s t test or Wilcoxon-Mann-Whitney test.

  • Fig. 6 TCS exposure increased basal inflammation through gut microbiota–dependent mechanisms.

    Conventionally raised and germ-free mice were treated with 80 ppm TCS in drinking water for 3 weeks. (A) Colon length. (B) Expression of Il-6 in colon. (C) Plasma concentration of IL-6. (D) Left: Colon histology (magnification, ×40). Right: Histological score. The results are expressed as means ± SEM. n = 5 to 12 per group. *P < 0.05, **P < 0.01, ****P < 0.0001. The statistical significance of the interaction effect between mouse type (conventionally raised versus germ-free mice) and treatment (TCS versus vehicle) on inflammation was determined by two-way ANOVA.

  • Fig. 7 Exposure to TCS activated TLR4 signaling in the DSS-induced colitis model.

    Mice were treated with 80 ppm TCS in diet for 3 weeks and then stimulated with DSS to induce colitis. (A) Effect of the plasma from the TCS- or vehicle-treated DSS mice on activation of TLR4, using a TLR4 reporter cell line (n = 6 to 9 per group). ABS, absorbance. (B) Plasma concentration of LPS from TCS- or vehicle-treated DSS mice (n = 6 per group). (C) qRT-PCR analysis of 16S rRNA gene in the blood and liver from TCS- or vehicle-treated DSS mice (n = 4 to 6 per group). (D) Plasma concentration of FITC-dextran (administered via oral gavage at 4 hours before euthanasia) from TCS- or vehicle-treated DSS mice (n = 4 to 5 per group). (E) qRT-PCR analysis of Occludin, Zo-1, Muc-3, and Tff-3 in colon of TCS- or vehicle-treated DSS mice (n = 6 to 11 per group). The results are expressed as means ± SEM. *P < 0.05, ***P < 0.001. The statistical significance was determined using Student’s t test or Wilcoxon-Mann-Whitney test.

  • Fig. 8 TCS enhanced DSS-induced colitis through a TLR4-dependent mechanism.

    WT and Tlr4−/− mice were treated with 80 ppm TCS in diet for 3 weeks and then stimulated with DSS to induce colitis. (A) Body weight. Left: Time course of mouse body weight after DSS treatment. Right: Quantification of mouse body weight on the day of euthanasia (day 6 after the initiation of DSS treatment). (B) Colon length. (C) FACS quantification of CD45+ cells in colon. (D) FACS quantification of CD45+ F4/80+ cells in colon. (E) Gene expression of Il-6 in colon. The results are expressed as means ± SEM. n = 6 to 10 per group. *P < 0.05, **P < 0.01. The statistical significance of the interaction effect between mouse type (Tlr4−/− versus WT mice) and treatment (TCS versus vehicle) on colitis was determined by two-way ANOVA.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/443/eaan4116/DC1

    Materials and Methods

    Fig. S1. LC-MS/MS analysis of TCS and its metabolite in mouse plasma.

    Fig. S2. Effects of TCS on cytokines in plasma.

    Fig. S3. Gating strategies used for analysis of major immune cell populations.

    Fig. S4. Effects of TCS on DSS-induced colitis in C57BL/6 mice.

    Fig. S5. Effects of TCS on colitis in Il-10−/− mice.

    Fig. S6. Effects of TCS (80 ppm in diet) on AOM/DSS-induced colon tumorigenesis in mice.

    Fig. S7. Effects of TCS (10 ppm in diet) on expression of PCNA and β-catenin in colon tumors.

    Fig. S8. Effects of TCS on fecal microbiota in mice.

    Fig. S9. Effects of TCS on B. infantis 272 growth.

    Table S1. Composition of diet used in the animal experiments.

    Table S2. Effects of TCS on composition of mouse microbiota at phylum levels.

    Table S3. Effects of TCS on composition of mouse microbiota at genus levels.

    Table S4. Sequences of primers used in qRT-PCR and 16S rRNA sequencing.

    Table S5. Primary data.

  • Supplementary Material for:

    A common antimicrobial additive increases colonic inflammation and colitis-associated colon tumorigenesis in mice

    Haixia Yang, Weicang Wang, Kymberleigh A. Romano, Min Gu, Katherine Z. Sanidad, Daeyoung Kim, Jun Yang, Birgitta Schmidt, Dipak Panigrahy, Ruisong Pei, Derek A. Martin, E. Ilker Ozay, Yuxin Wang, Mingyue Song, Bradley W. Bolling, Hang Xiao, Lisa M. Minter, Guang-Yu Yang, Zhenhua Liu, Federico E. Rey,* Guodong Zhang*

    *Corresponding author. Email: guodongzhang{at}umass.edu (G.Z.); ferey{at}wisc.edu (F.E.R.)

    Published 30 May 2018, Sci. Transl. Med. 10, eaan4116 (2018)
    DOI: 10.1126/scitranslmed.aan4116

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. LC-MS/MS analysis of TCS and its metabolite in mouse plasma.
    • Fig. S2. Effects of TCS on cytokines in plasma.
    • Fig. S3. Gating strategies used for analysis of major immune cell populations.
    • Fig. S4. Effects of TCS on DSS-induced colitis in C57BL/6 mice.
    • Fig. S5. Effects of TCS on colitis in Il-10−/− mice.
    • Fig. S6. Effects of TCS (80 ppm in diet) on AOM/DSS-induced colon tumorigenesis in mice.
    • Fig. S7. Effects of TCS (10 ppm in diet) on expression of PCNA and β-catenin in colon tumors.
    • Fig. S8. Effects of TCS on fecal microbiota in mice.
    • Fig. S9. Effects of TCS on B. infantis 272 growth.
    • Table S1. Composition of diet used in the animal experiments.
    • Table S2. Effects of TCS on composition of mouse microbiota at phylum levels.
    • Table S3. Effects of TCS on composition of mouse microbiota at genus levels.
    • Table S4. Sequences of primers used in qRT-PCR and 16S rRNA sequencing.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S5 (Microsoft Excel format). Primary data.

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