Dietary emulsifiers impact the mouse gut microbiota promoting colitis and metabolic syndrome (original) (raw)

Accession codes

Primary accessions

European Nucleotide Archive

Data deposits

Sequencing data are deposited in the European Nucleotide Archive under accession number PRJEB8035.

References

  1. Johansson, M. E. et al. The inner of the two Muc2 mucin-dependent mucus layers in colon is devoid of bacteria. Proc. Natl Acad. Sci. USA 105, 15064–15069 (2008)
    ADS CAS PubMed PubMed Central Google Scholar
  2. Roberts, C. L. et al. Translocation of Crohn’s disease Escherichia coli across M-cells: contrasting effects of soluble plant fibres and emulsifiers. Gut 59, 1331–1339 (2010)
    PubMed Google Scholar
  3. Swidsinski, A., Loening-Baucke, V. & Herber, A. Mucosal flora in Crohn’s disease and ulcerative colitis - an overview. J. Physiol. Pharmacol. 60 (Suppl 6). 61–71 (2009)
    PubMed Google Scholar
  4. Food Safety Commission [of Japan]. Evaluation Report of Food Additives (Polysorbates 20, 60, 65 and 80) (2007) [transl.] https://www.fsc.go.jp/english/evaluationreports/foodadditive/polysorbate_report.pdf
  5. National Toxicology Program Toxicology and Carcinogenesis Studies of Polysorbate 80 (CAS No. 9005–65–6) in F344/N Rats and B6C3F1 Mice (Feed Studies). Natl. Toxicol. Program Tech. Rep. Ser. 415, 1–225 (1992)
    Google Scholar
  6. Swidsinski, A. et al. Bacterial overgrowth and inflammation of small intestine after carboxymethylcellulose ingestion in genetically susceptible mice. Inflamm. Bowel Dis. 15, 359–364 (2009)
    PubMed Google Scholar
  7. Kühn, R., Lohler, J., Rennick, D., Rajewsky, K. & Muller, W. Interleukin-10-deficient mice develop chronic enterocolitis. Cell 75, 263–274 (1993)
    PubMed Google Scholar
  8. Vijay-Kumar, M. et al. Deletion of TLR5 results in spontaneous colitis in mice. J. Clin. Invest. 117, 3909–3921 (2007)
    CAS PubMed PubMed Central Google Scholar
  9. Ubeda, C. et al. Familial transmission rather than defective innate immunity shapes the distinct intestinal microbiota of TLR-deficient mice. J. Exp. Med. 209, 1445–1456 (2012)
    CAS PubMed PubMed Central Google Scholar
  10. Chassaing, B., Ley, R. E. & Gewirtz, A. T. Intestinal epithelial cell Toll-like receptor 5 regulates the intestinal microbiota to prevent low-grade inflammation and metabolic syndrome in mice. Gastroenterology 147, 1363–1377 (2014)
    CAS PubMed Google Scholar
  11. Johansson, M. E. & Hansson, G. C. Preservation of mucus in histological sections, immunostaining of mucins in fixed tissue, and localization of bacteria with FISH. Methods Mol. Biol. 842, 229–235 (2012)
    CAS PubMed Google Scholar
  12. Lozupone, C. & Knight, R. UniFrac: a new phylogenetic method for comparing microbial communities. Appl. Environ. Microbiol. 71, 8228–8235 (2005)
    CAS PubMed PubMed Central Google Scholar
  13. Bäckhed, F., Ley, R. E., Sonnenburg, J. L., Peterson, D. A. & Gordon, J. I. Host-bacterial mutualism in the human intestine. Science 307, 1915–1920 (2005)
    ADS PubMed Google Scholar
  14. Turnbaugh, P. J. et al. An obesity-associated gut microbiome with increased capacity for energy harvest. Nature 444, 1027–1031 (2006)
    ADS PubMed Google Scholar
  15. Qin, J. et al. A human gut microbial gene catalogue established by metagenomic sequencing. Nature 464, 59–65 (2010)
    CAS PubMed PubMed Central Google Scholar
  16. Png, C. W. et al. Mucolytic bacteria with increased prevalence in IBD mucosa augment in vitro utilization of mucin by other bacteria. Am. J. Gastroenterol. 105, 2420–2428 (2010)
    ADS CAS PubMed Google Scholar
  17. Arthur, J. C. et al. Intestinal inflammation targets cancer-inducing activity of the microbiota. Science 338, 120–123 (2012)
    ADS CAS PubMed PubMed Central Google Scholar
  18. Carvalho, F. A. et al. Transient inability to manage proteobacteria promotes chronic gut inflammation in TLR5-deficient mice. Cell Host Microbe 12, 139–152 (2012)
    CAS PubMed PubMed Central Google Scholar
  19. Mukhopadhya, I., Hansen, R., El-Omar, E. M. & Hold, G. L. IBD-what role do Proteobacteria play? Nature Rev. Gastroenterol. Hepatol. 9, 219–230 (2012)
    CAS Google Scholar
  20. Cox, L. M. et al. Altering the intestinal microbiota during a critical developmental window has lasting metabolic consequences. Cell 158, 705–721 (2014)
    CAS PubMed PubMed Central Google Scholar
  21. Chassaing, B., Koren, O., Carvalho, F. A., Ley, R. E. & Gewirtz, A. T. AIEC pathobiont instigates chronic colitis in susceptible hosts by altering microbiota composition. Gut 63, 1069–1080 (2014)
    CAS PubMed Google Scholar
  22. Ziegler, T. R. et al. Detectable serum flagellin and lipopolysaccharide and upregulated anti-flagellin and lipopolysaccharide immunoglobulins in human short bowel syndrome. Am. J. Physiol. Regul. Integr. Comp. Physiol. 294, R402–R410 (2008)
    CAS PubMed Google Scholar
  23. Chassaing, B. et al. Fecal lipocalin 2, a sensitive and broadly dynamic non-invasive biomarker for intestinal inflammation. PLoS ONE 7, e44328 (2012)
    ADS CAS PubMed PubMed Central Google Scholar
  24. Devkota, S. et al. Dietary-fat-induced taurocholic acid promotes pathobiont expansion and colitis in _Il10_−/− mice. Nature 487, 104–108 (2012)
    ADS CAS PubMed PubMed Central Google Scholar
  25. Kullberg, M. C. et al. Helicobacter hepaticus triggers colitis in specific-pathogen-free interleukin-10 (IL-10)-deficient mice through an IL-12- and gamma interferon-dependent mechanism. Infect. Immun. 66, 5157–5166 (1998)
    CAS PubMed PubMed Central Google Scholar
  26. Gregor, M. F. & Hotamisligil, G. S. Inflammatory mechanisms in obesity. Annu. Rev. Immunol. 29, 415–445 (2011)
    CAS PubMed Google Scholar
  27. Vijay-Kumar, M. et al. Metabolic syndrome and altered gut microbiota in mice lacking Toll-like receptor 5. Science 328, 228–231 (2010)
    ADS CAS PubMed PubMed Central Google Scholar
  28. Wong, T. et al. Divergent metabolic adaptations to intestinal parasitic nematode infection in mice susceptible or resistant to obesity. Gastroenterology 133, 1979–1988 (2007)
    CAS PubMed Google Scholar
  29. Furusawa, Y. et al. Commensal microbe-derived butyrate induces the differentiation of colonic regulatory T cells. Nature 504, 446–450 (2013)
    ADS CAS PubMed Google Scholar
  30. Suez, J. et al. Artificial sweeteners induce glucose intolerance by altering the gut microbiota. Nature 514, 181–186 (2014)
    ADS CAS PubMed Google Scholar
  31. Uematsu, S. et al. Detection of pathogenic intestinal bacteria by Toll-like receptor 5 on intestinal CD11c+ lamina propria cells. Nature Immunol. 7, 868–874 (2006)
    CAS Google Scholar
  32. Denizot, J. et al. Adherent-invasive Escherichia coli induce claudin-2 expression and barrier defect in CEABAC10 mice and Crohn’s disease patients. Inflamm. Bowel Dis. 18, 294–304 (2012)
    PubMed Google Scholar
  33. Castaneda, F. E. et al. Targeted deletion of metalloproteinase 9 attenuates experimental colitis in mice: central role of epithelial-derived MMP. Gastroenterology 129, 1991–2008 (2005)
    CAS PubMed Google Scholar
  34. Katakura, K. et al. Toll-like receptor 9-induced type I IFN protects mice from experimental colitis. J. Clin. Invest. 115, 695–702 (2005)
    CAS PubMed PubMed Central Google Scholar
  35. Sitaraman, S. V. et al. Elevated flagellin-specific immunoglobulins in Crohn’s disease. Am. J. Physiol. Gastrointest. Liver Physiol. 288, G403–G406 (2005)
    CAS PubMed Google Scholar
  36. Turnbaugh, P. J. et al. A core gut microbiome in obese and lean twins. Nature 457, 480–484 (2009)
    ADS CAS PubMed Google Scholar
  37. Gilbert, J. A. et al. The Earth Microbiome Project: Meeting report of the “1 EMP meeting on sample selection and acquisition” at Argonne National Laboratory October 6 2010. Stand. Genomic Sci. 3, 249–253 (2010)
    MathSciNet PubMed PubMed Central Google Scholar
  38. Caporaso, J. G. et al. Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. ISME J. 6, 1621–1624 (2012)
    CAS PubMed PubMed Central Google Scholar
  39. Caporaso, J. G. et al. QIIME allows analysis of high-throughput community sequencing data. Nature Methods 7, 335–336 (2010)
    CAS PubMed PubMed Central Google Scholar
  40. Aronesty, E. Comparison of sequencing utility programs. Open Bioinformatics J. 7, 1–8 (2013)
    MathSciNet Google Scholar
  41. Edgar, R. C. Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26, 2460–2461 (2010)
    CAS PubMed Google Scholar
  42. McDonald, D. et al. An improved Greengenes taxonomy with explicit ranks for ecological and evolutionary analyses of bacteria and archaea. ISME J. 6, 610–618 (2012)
    CAS PubMed Google Scholar
  43. Price, M. N., Dehal, P. S. & Arkin, A. P. FastTree: computing large minimum evolution trees with profiles instead of a distance matrix. Mol. Biol. Evol. 26, 1641–1650 (2009)
    CAS PubMed PubMed Central Google Scholar
  44. Lozupone, C., Hamady, M. & Knight, R. UniFrac–an online tool for comparing microbial community diversity in a phylogenetic context. BMC Bioinformatics 7, 371 (2006)
    PubMed PubMed Central Google Scholar
  45. Tibshirani, R., Hastie, T., Narasimhan, B. & Chu, G. Diagnosis of multiple cancer types by shrunken centroids of gene expression. Proc. Natl Acad. Sci. USA 99, 6567–6572 (2002)
    ADS CAS PubMed PubMed Central Google Scholar

Download references

Acknowledgements

This work was supported by NIH grant DK099071 and DK083890. B.C. is a recipient of the Research Fellowship award from the Crohn’s and Colitis Foundation of America (CCFA). We thank B. Zhang, L. Etienne-Mesmin, H. Q. Tran and E. Viennois for technical assistance.

Author information

Authors and Affiliations

  1. Center for Inflammation, Immunity and Infection, Institute for Biomedical Sciences, Georgia State University, Atlanta, 30303, Georgia, USA
    Benoit Chassaing & Andrew T. Gewirtz
  2. Faculty of Medicine, Bar Ilan University, Safed, 13115, Israel,
    Omry Koren
  3. Department of Molecular Biology and Genetics, Cornell University, Ithaca, 14853, New York, USA
    Julia K. Goodrich, Angela C. Poole & Ruth E. Ley
  4. Digestive Diseases Division, Department of Medicine, Emory University School of Medicine, Atlanta, 30322, Georgia, USA
    Shanthi Srinivasan

Authors

  1. Benoit Chassaing
    You can also search for this author inPubMed Google Scholar
  2. Omry Koren
    You can also search for this author inPubMed Google Scholar
  3. Julia K. Goodrich
    You can also search for this author inPubMed Google Scholar
  4. Angela C. Poole
    You can also search for this author inPubMed Google Scholar
  5. Shanthi Srinivasan
    You can also search for this author inPubMed Google Scholar
  6. Ruth E. Ley
    You can also search for this author inPubMed Google Scholar
  7. Andrew T. Gewirtz
    You can also search for this author inPubMed Google Scholar

Contributions

B.C. and A.T.G. conceived the project, designed the experiments, interpreted the results, and wrote the manuscript. B.C. performed all experiments and analysis with advice and guidance from O.K., J.K.G., and A.C.P. S.S. and R.E.L. guided experimental design and data interpretation.

Corresponding author

Correspondence toAndrew T. Gewirtz.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Effects of emulsifiers on mucus-microbiota interaction in wild-type, _Il10_−/− and _Tlr5_−/− mice.

a–d, Dietary emulsifiers did not affect mucus and mucus-related genes expression in wild-type mice. Wild-type (WT) mice were exposed to drinking water containing CMC or P80 (1.0%) for 12 weeks. a–c, mRNA expression analysis by qRT–PCR of Muc2 (a), Tff3 (b) and Klf4 (c) genes in the colonic mucosa. Points are from individual mice, bar represent the mean ± s.e.m., (n = 9). d, Colons were stained using periodic acid–Schiff stains. Scale bar, 200 μm. Pictures are representative of 10 biological replicates. e–j, Dietary emulsifiers alter microbiota localization, composition and pro-inflammatory potential in _Tlr5_−/− mice. _Tlr5_−/− mice were exposed to drinking water containing CMC or P80 (1.0%) for 12 weeks. e–g, Confocal microscopy analysis of microbiota localization: MUC2, green; actin, purple; bacteria, red; and DNA, blue. Scale bar, 20 μm. Pictures are representative of five biological replicates. h, Distances of closest bacteria to intestinal epithelial cells (IEC) per condition over five high-powered fields per mouse. i, j, PCR-based quantification of total bacterial load (i) and bacterial load adhered to colonic mucosa (j). Data are the means ± s.e.m., n = 5. Significance was determined using one-way ANOVA corrected for multiple comparisons with a Sidak test; P < 0.05 compared to water-treated group. k, l, Dietary emulsifiers do not modify total bacterial load in wild-type and _Il10_−/− mice. Wild-type and _Il10_−/− mice were exposed to drinking water containing CMC or P80 (1.0%) for 12 weeks. Total bacterial load in stool of wild-type (k) and _Il10_−/− (l) mice. Points are from individual mice. Data are geometric means with 95% confidence interval (n = 5 for k except n = 4 for CMC- and P80-treated groups; for l, n = 8, 4 and 6 for water-, CMC- and P80-treated groups, respectively).

Extended Data Figure 2 Emulsifiers alter microbiota composition.

a–h, Dietary emulsifiers induce profound alterations in gut microbiota composition in wild-type and _Il10_−/− mice. Wild-type and _Il10_−/− mice were exposed to drinking water containing CMC or P80 (1.0%) for 12 weeks. a, b, e, f, Day 0 (a, e) and day 93 (b, f) microbiota richness and diversity in wild-type (a, b) and _Il10_−/− (e, f). c, d, g, h, Principal coordinates analysis (PCoA) of the unweighted UniFrac distance matrix of faecal microbiota (c, g) and mucosa-associated bacteria (d, h) in wild-type (c, d) and _Il10_−/− (g, h) mice. Treatment of each mouse is indicated by point colour and matching coloured circles represent clustering by treatment (blue, water; orange, CMC; purple, P80). Black dashed circles represent mice sharing a cage. Data are the means ± s.e.m.; n = 5, except n = 4 for P80-treated wild-type mice; n = 4 for CMC-treated _Il10_−/− mice; and n = 9 for water-treated _Il10_−/− mice. Significance was determined using two-way group ANOVA (#P < 0.05) compared to the water-treated group. i, j, Phylum characterization of emulsifier-induced alteration of gut microbiota composition in wild-type and _Il10_−/− mice. Wild-type and _Il10_−/− mice were exposed to drinking water containing CMC or P80 (1.0%) for 12 weeks. Relative abundance of phyla are represented for faecal microbiota at day 93 (i) and for colonic mucosa-associated bacteria (j). Data are the means ± s.e.m., n = 5. Significance was determined using two-way ANOVA corrected for multiple comparisons with a Bonferroni test, P < 0.05 compared to water-treated group. k–o, Dietary emulsifiers induce profound alterations in gut microbiota composition in _Tlr5_−/− mice. _Tlr5_−/− mice were exposed to drinking water containing CMC or P80 (1.0%) for 12 weeks. k, l, Day 0 (k) and day 93 (l) microbiota richness and diversity (n = 5). m–o, PCoA of the unweighted UniFrac distance matrix of faecal microbiota at day 0 (m), day 93 (n) and of mucosa-associated bacteria (o). Data are the means ± s.e.m. (for m, n = 4, 5 and 5 for water-, CMC- and P80-treated groups, respectively; for n, n = 4, 5 and 3 for water-, CMC- and P80-treated groups, respectively; for o, n = 4). Significance was determined using two-way group ANOVA (#P < 0.05) compared to the water-treated group. p–t, Prevalence analysis of OTUs related to mucolytic bacteria in _Il10_−/− mice treated with dietary emulsifier. _Il10_−/− mice were exposed to drinking water containing CMC or P80 (1.0%) for 12 weeks. OTUs Prok_MSA # 52947 (p; related to Clostridium perfringens), 264696 (q; related to Akkermansia muciniphila), 315982 (r; related to Clostridium perfringens), 363731 (s; related to Akkermansia muciniphila), and 178331(t; related to Akkermansia muciniphila) were analysed. Data are expressed as a percentage of the total sequences analysed and are the means ± s.e.m. (n = 6, except n = 9 for water-treated _Il10_−/− mice). Significance was determined using two-way ANOVA corrected for multiple comparisons with a Bonferroni test, P < 0.05 compared to water-treated group.

Extended Data Figure 3 Emulsifier effects on microbiota are irrespective of cage clustering.

a–g, Dietary emulsifiers alter gut microbiota composition in wild-type mice relative to littermate controls. a–d, All the female (n = 33; a, b) and male (n = 33; c, d) mice from 10 different litters were placed into cages in a manner such that each litter was split equally amongst the three experimental groups that were to receive water, CMC or P80 (three cages per sex per condition). Mice were exposed to drinking water containing CMC or P80 (1.0%) for 8 weeks. a–d, Day 0 (a, c) and day 63 (b, d) Principal coordinates analysis (PCoA) of the unweighted UniFrac distance matrix of faecal microbiota in wild-type mice. Treatment of each mouse is indicated by point colour (blue, water; orange, CMC; purple, P80). The mother of each mouse is indicated by point colour. The cage of each mouse is indicated by point colour. e, f, Mice were clustered using UPGMA (unweighted pair group method with arithmetic mean). Treatment of each mouse is indicated by line colour (blue, water; orange, CMC; purple, P80). g, Schematic representation of the above experimental design. h–n, Dietary emulsifiers alter gut microbiota composition in _Tlr5_−/− mice relative to littermate controls. All the female (n = 25; h, i) and male (n = 23; j, k) mice from eight different litters were placed into cages in a manner such that each litter was split equally amongst the three experimental groups that were to receive water, CMC or P80 (two cages per sex per condition). Mice were exposed to drinking water containing CMC or P80 (1.0%) for 8 weeks. h–k, Day 0 (h, j) and day 63 (i, k) PCoA of the unweighted UniFrac distance matrix of faecal microbiota in _Tlr5_−/− mice. Treatment of each mouse is indicated by point colour (blue, water; orange, CMC; purple, P80). The mother of each mouse is indicated by point colour. The cage of each mouse is indicated by point colour. l, m, Mice were clustered using UPGMA (unweighted pair group method with arithmetic mean). Treatment of each mouse is indicated by line colour (blue = water; orange = CMC; purple = P80). n, Schematic representation of the above experimental design.

Extended Data Figure 4 OTUs altered by emulsifiers.

a–d, Misclassification error rate and heat map representation of the 15 most significantly altered OTUs in wild-type mice treated with dietary emulsifier. a–d, Wild-type mice were exposed to drinking water containing CMC (a, b) or P80 (c, d) (1.0%) for 12 weeks. a, c, Misclassification error rate showing that 15 OTUs were sufficient to successfully discriminate microbiota from each experimental group (error rate = 0). b, d, Heat map representation of the 15 most significantly altered OTUs in wild-type mice treated with dietary emulsifiers. Colours represent relative expression (white and red for underrepresented and overrepresented, respectively). The 15 OTUs are listed on the right using their Greengenes Prok_MSA identities, and assigned taxonomy are labelled starting phylum, then class, order, family and genus. Dendrogram on the upper part represents sample clustering. e–h, As for a–d with _Il10_−/− mice. i–l, As for a–d with _Tlr5_−/− mice. For a, b, n = 5; for c, d, n = 5 and 4 for water- and P80-treated groups, respectively; for e, f, n = 5 and 4 for water- and CMC-treated groups, respectively; for g, h, n = 5 and 4 for water- and P80-treated groups, respectively; for i, j, n = 4; for k, l, n = 4 and 3 for water- and P80-treated groups, respectively.

Extended Data Figure 5 Emulsifier-induced changes in adult mouse microbiota.

a–g, Dietary emulsifiers promote metabolic syndrome in adult wild-type mice. a–g, Four-month-old male wild-type (WT) mice were exposed to drinking water containing CMC or P80 (1.0%) for 8 weeks (two cages per condition). a, Body weight over time; b, 15 h fasting blood glucose concentration; c, food intake measurement; d, spleen weights; e, fat-pad weights; f, colon weights; and g, colon lengths. Data are the means ± s.e.m., n = 10. Significance was determined using one-way ANOVA corrected for multiple comparisons with a Sidak test (P < 0.05 compared to water-treated group) or two-way group ANOVA (#P < 0.05 compared to water-treated group). h–p, Dietary emulsifiers alter gut microbiota composition in adult wild-type mice. Four-month-old male wild-type mice were exposed to drinking water containing CMC or P80 (1.0%) for 8 weeks (two cages per condition). h, i, Day 0 (h) and day 63 (i) Principal coordinates analysis (PCoA) of the unweighted UniFrac distance matrix of faecal microbiota in wild-type mice. Treatment of each mouse is indicated by point colour (blue, water; orange, CMC; purple, P80). The cage of each mouse is indicated by point colour (for h, n = 7, 8 and 8 for water-, CMC- and P80-treated groups, respectively). j, Day 0 and day 63 microbiota richness and diversity. k, Day 63 jackknifed PCoA of the unweighted UniFrac distance matrix of faecal microbiota in wild-type mice. Treatment of each mouse is indicated by point colour (blue, water; orange, CMC; purple, P80) (n = 8). l, After clustering of mouse faecal microbiota using PCoA of the unweighted UniFrac distance matrix, a representative mouse has been used to illustrate the time point evolution of the microbiota (n = 1). m, After clustering of mouse faecal microbiota using PCoA of the unweighted UniFrac distance matrix, evolution of the principal coordinate 1 between day 0 and day 63 has been calculated for each mouse (n = 10). n, Average of the UniFrac unweighted distance for each group (water, CMC and P80) between day 0 and day 63 has been calculated (n = 10). o, Average of the UniFrac unweighted distance within group (water, CMC and P80) has been calculated (n = 10). p, Average of the UniFrac unweighted distance between group (cages, water, CMC and P80) or within group (water) at day 63 has been calculated (n = 10). Significance was determined using one-way ANOVA corrected for multiple comparisons with a Sidak test (P < 0.05 compared to water-treated group) or two-way group ANOVA (#P < 0.05 compared to water-treated group). q, r, Dietary emulsifiers increase pro-inflammatory potential of intestinal microbiota in _Tlr5_−/− mice. _Tlr5_−/− mice were exposed to drinking water containing CMC or P80 (1.0%) for 12 weeks. Bioactive levels of faecal flagellin (q) and LPS (r) assayed with TLR5 and TLR4 reporter cells. Data are the means ± s.e.m., n = 10. Significance was determined using two-way ANOVA corrected for multiple comparisons with a Bonferroni test; P < 0.05 compared to water-treated group. s–v, Dietary emulsifiers increase serum immune reactivity. Wild-type (s, t) and _Il10_−/− (u, v) mice were exposed to drinking water containing CMC or P80 (1.0%) for 12 weeks. Serum immune reactivity (IgG) to flagellin (s, u) and LPS (t, v) in wild-type (s, t) and _Il10_−/− (u, v) mice. Points are from individual mice. Data are the means ± s.e.m. (for s, n = 18, 30 and 16 for water-, CMC- and P80-treated groups, respectively; for t, n = 18, 31 and 17 for water-, CMC- and P80-treated groups, respectively; for u, n = 21, 20 and 23 for water-, CMC- and P80-treated groups, respectively; for v, n = 27, 20 and 25 for water-, CMC- and P80-treated groups, respectively). Significance was determined using one-way ANOVA corrected for multiple comparisons with a Sidak test; P < 0.05 compared to water-treated group.

Extended Data Figure 6 Histopathologic changes in emulsifier-treated wild-type and Il10 −/− mice.

a, Dietary emulsifiers induce histopathologically robust inflammation in _Il10_−/− mice. a, _Il10_−/− mice were exposed to drinking water containing CMC or P80 (1.0%) for 12 weeks. Colon and small intestine were haematoxylin and eosin (H&E) stained. Scale bar, 200 μm. Pictures are representatives of 15 biological replicates. b, Dietary emulsifiers induce histopathologically robust inflammation in _Tlr5_−/− mice. _Tlr5_−/− mice were exposed to drinking water containing CMC or P80 (1.0%) for 12 weeks. Colon and small intestine were H&E stained. Scale bar, 200 μm. Pictures are representative of five biological replicates. c, Histopathology of emulsifier-treated wild-type (WT) mice. Wild-type mice were exposed to drinking water containing CMC or P80 (1.0%) for 12 weeks. Colon and small intestine were H&E stained. Scale bar, 200 μm. Pictures are representative of 15 biological replicates. d–h, Dietary emulsifiers elicit low-grade intestinal inflammation in WT and splenomegaly in _Il10_−/− mice. Wild-type (d–g) and _Il10_−/− (h) mice were exposed to drinking water containing CMC or P80 (1.0%) for 12 weeks. d, Colitis incidence over time; e, h, spleen weights; f, epithelial damage; and g, infiltration scores. Points are from individual mice, bars represent the mean. For e–g, n = 14, 27 and 16 for water-, CMC- and P80-treated groups, respectively; for h, n = 11, 18 and 20 for water-, CMC- and P80-treated groups, respectively. Significance was determined using one-way ANOVA corrected for multiple comparisons with a Sidak test; P < 0.05 compared to water-treated group. i–k, Extent of intestinal inflammation correlates with perturbation in microbiota localization in wild-type and _Il10_−/− mice. _Il10_−/− mice were exposed to drinking water containing CMC (i) or P80 (j) (1.0%) for 12 weeks. Faecal levels of the inflammatory marker LCN2 as well as confocal microscopy analysis of microbiota localization and estimation of the distances of the closest bacteria to intestinal epithelial cells (IEC) were determined, and plotted in the x and y axis, respectively. Linear regression line was calculated and _R_2 was determined; n = 11. k, Analysis of bacterial–epithelial distance upon stratification of levels of gut inflammatory marker faecal LCN2, using both wild-type and _Il10_−/− mice exposed to drinking water containing CMC or P80 (1.0%) for 12 weeks. Mice were grouped according to their faecal LCN2 levels and bacterial–epithelial distances were then plotted (mean ± s.e.m.). Significance was determined using one-way ANOVA corrected for multiple comparisons with a Sidak test; P < 0.05 compared to X < 50 ng per g group.

Extended Data Figure 7 Inflammatory and metabolic parameters in emulsifier-treated wild-type and Tlr5 −/− mice.

a–g, Dietary emulsifiers promote intestinal inflammation in _Tlr5_−/− mice. a–g, _Tlr5_−/− mice were exposed to drinking water containing CMC or P80 (1.0%) for 12 weeks. a, Faecal levels of the inflammatory marker LCN2 over time; b, colitis incidence over time; c, myeloperoxidase levels; d, histological score; e, colon weights; f, colon lengths; and g, spleen weights. Data are the means ± s.e.m. or geometric means with 95% confidence interval (the latter for a), n = 5. Significance was determined using one-way ANOVA corrected for multiple comparisons with a Sidak test or using two-way ANOVA corrected for multiple comparisons with a Bonferroni test; P < 0.05 compared to water-treated group. h–k, Dietary emulsifiers induce metabolic syndrome in wild-type (WT) and _Tlr5_−/− mice. Wild-type (h, i) and _Tlr5_−/− (j, k) mice were exposed to drinking water containing CMC or P80 (1.0%) for 12 weeks. Glucose tolerance (h, j) and insulin sensitivity (i, k) were analysed. Data are the means ± s.e.m., n = 5. Significance was determined using two-way group ANOVA (#P < 0.05) compared to water-treated group. l–t, Emulsifier-supplemented chow elicits low-grade intestinal inflammation in wild-type mice. Wild-type mice were given mouse chow containing CMC or P80 (1.0%) for 12 weeks. l, Faecal levels of the inflammatory marker LCN2 over time; m, myeloperoxidase levels; n, food intake measurement; o, 15 h fasting blood glucose concentration; p, colon weights; q, colon lengths; r, spleen weights; and s, t, PCR-based quantification of total bacterial load (s) and bacterial load adhered to colonic mucosa (t). Data are the means ± s.e.m. or geometric means with 95% confidence interval (the latter for l), n = 5. Significance was determined using one-way ANOVA corrected for multiple comparisons with a Sidak test or using two-way ANOVA corrected for multiple comparisons with a Bonferroni test; P < 0.05 compared to control group. u–h′ Dose-response characterization of dietary emulsifiers on intestinal inflammation. Wild-type mice were exposed to drinking water containing 0.1–1.0% CMC (u–a′) or P80 (b′–h′) for 12 weeks. u, b′, Faecal levels of the inflammatory marker LCN2 over time; v, c′, myeloperoxidase levels; w, d′, food intake measurement; x, e′, 15 h fasting blood glucose concentration; y, f′, colon weights; z, g′, colon lengths; and a′, h′, spleen weights. Data are the means ± s.e.m., n = 5. Significance was determined using one-way ANOVA corrected for multiple comparisons with a Sidak test or using two-way ANOVA corrected for multiple comparisons with a Bonferroni test; P < 0.05 compared to water-treated group.

Extended Data Figure 8 Reversibility and dose dependence of emulsifier-induced effects on inflammation and metabolism.

a–k, Emulsifier-induced metabolic syndrome in Swiss Webster mice is partially reversible by 6 weeks after emulsifier treatment. a, Schematic representation of the experiment. b, c, Body weight over time; d, fat-pad weight; e, faecal levels of the inflammatory marker LCN2 over time; f, myeloperoxidase levels; g, food intake measurement; h, 15 h fasting blood glucose concentration; i, colon weights; j, colon lengths; and k, spleen weights. Data are the means ± s.e.m., n = 5. Significance was determined using one-way ANOVA corrected for multiple comparisons with a Sidak test (P < 0.05 compared to water-treated group) or two-way group ANOVA (#P < 0.05 compared to water-treated group). l–s, Sodium sulfite did not induce robust or low-grade intestinal inflammation. Wild-type and _Il10_−/− mice were exposed to drinking water containing sodium sulfite (1.0%) for 12 weeks. l, m, Body weight over time; n, colon weights; o, colon lengths; p, spleen weights; q, fat-pad weight; r, myeloperoxidase levels; and s, serum levels of the inflammatory marker LCN2. Data are the means ± s.e.m., n = 5. Points are from individual mice. Significance was determined using one-way ANOVA corrected for multiple comparisons with a Sidak test. t–w, Dietary emulsifiers promote metabolic syndrome in _Tlr5_−/− mice. _Tlr5_−/− mice were exposed to drinking water containing CMC or P80 (1.0%) for 12 weeks. t, Body weight over time; u, fat-pad weight; v, food intake measurement; and w, 15 h fasting blood glucose concentration. Data are the means ± s.e.m., n = 5. Significance was determined using one-way ANOVA corrected for multiple comparisons with a Sidak test (P < 0.05 compared to water-treated group) or two-way group ANOVA (#P < 0.05 compared to water-treated group). x–f′, Emulsifier-supplemented chow promotes intestinal inflammation in _Tlr5_−/− mice. _Tlr5_−/− mice were given mouse chow containing CMC or P80 (1.0%) for 12 weeks. x, Faecal levels of the inflammatory marker LCN2 over time; y, myeloperoxidase levels; z, food intake measurement; a′, 15 h fasting blood glucose concentration; b′, colon weights; c′, colon lengths; d′, spleen weights; and e′, f′, PCR-based quantification of total bacterial load (e′) and bacterial load adhered to colonic mucosa (f′). Data are the means ± s.e.m., n = 5. Significance was determined using one-way ANOVA corrected for multiple comparisons with a Sidak test or using two-way ANOVA corrected for multiple comparisons with a Bonferroni test; P < 0.05 compared to control group. g′–o′, Dose-response characterization of dietary emulsifiers on intestinal inflammation in _Tlr5_−/− mice. _Tlr5_−/− mice were exposed to drinking water containing 0.1–1.0% P80 for 12 weeks. g′, Body weight over time; h′, fat-pad weight; i′, faecal levels of the inflammatory marker LCN2 over time; j′, myeloperoxidase levels; k′, food intake measurement; l′, 15 h fasting blood glucose concentration; m′, colon weights; n′, colon lengths; and o′, spleen weights. Data are the means ± s.e.m., n = 3. Points in d are from individual mice. Significance was determined using one-way ANOVA corrected for multiple comparisons with a Sidak test (P < 0.05 compared to water-treated group) or two-way group ANOVA (#P < 0.05 compared to water-treated group).

Extended Data Figure 9 Effects of emulsifiers on metabolic parameters and bile acids in conventional and germ-free mice.

a–f, Dietary emulsifiers promotes metabolic syndrome in _Il10_−/− mice. _Il10_−/− mice were exposed to drinking water containing CMC or P80 (1.0%) for 12 weeks. a–c, Body weight over time; d, fat-pad weight; e, food intake measurement; and f, 15 h fasting blood glucose concentration. Data are the means ± s.e.m. (for a, n = 24, 18 and 21 for water-, CMC- and P80-treated groups, respectively; for b, n = 14, 11 and 8 for water-, CMC- and P80-treated groups, respectively; for c, n = 14, 9 and 9 for water-, CMC- and P80-treated groups, respectively; for d, n = 14, 18 and 20 for water-, CMC- and P80-treated groups, respectively; for e, n = 15, 11 and 12 for water-, CMC- and P80-treated groups, respectively; for f, n = 21, 17 and 20 for water-, CMC- and P80-treated groups, respectively). Significance was determined using one-way ANOVA corrected for multiple comparisons with a Sidak test (P < 0.05 compared to water-treated group) or two-way group ANOVA (#P < 0.05 compared to water-treated group). Points are from individual mice and red points in f represent mice with overt colitis. g–p, Emulsifier-induced low-grade intestinal inflammation was abolished under germ-free conditions. Conventionally housed (g–k) and germ-free (l–p) Swiss Webster mice were exposed to drinking water containing CMC or P80 (1.0%) for 12 weeks. g, l, Faecal levels of the inflammatory marker LCN2 over time; h, m, myeloperoxidase levels; i, n, colon weights; j, o, colon lengths; and k, p, spleen weights. Data are the means ± s.e.m.; n = 8 for conventionally housed mice and n = 4 for germ-free mice. Significance was determined using one-way ANOVA corrected for multiple comparisons with a Sidak test or using two-way ANOVA corrected for multiple comparisons with a Bonferroni test, P < 0.05 compared to control group. q–w, Dietary emulsifiers induce perturbations in faecal short-chain fatty acid composition. Faecal short-chain fatty acids composition was analysed at the Metabolomics Core of the University of Michigan. Acetate (q), propionate (r), butyrate (s), isovalerate (t), valerate (u), hexanoate (v) and heptanoate (w) were analysed. Data are the means ± s.e.m. (n = 5, 8 and 7 for water-, CMC- and P80-treated conventional mice groups, respectively; n = 4, 4 and 5 for water-, CMC- and P80-treated germ-free mice groups, respectively). Significance was determined using one-way ANOVA corrected for multiple comparisons with a Sidak test; P < 0.05 compared to water-treated group. x–a′, Dietary emulsifiers promote metabolic syndrome in mice from different vendors. Wild-type mice were exposed to drinking water containing CMC or P80 (1.0%) for 12 weeks. x, y, Body weight over time of Bl/6 mice used upon receipt from Jackson Laboratories (x) or bred at Georgia State University (y). z, a′, Body weight over time of Swiss Webster mice used upon receipt from Charles River company (z) or bred at Georgia State University (a′). Data in x are not used elsewhere in report, while data in y, z, and a′ are from Figs 3a and 4a and Extended Data Fig 8a–k. Data are the means ± s.e.m. n = 8 for Bl/6 mice used upon receipt from Jackson Laboratories, n = 16 for Bl/6 mice bred at Georgia State University, n = 10 for Swiss Webster mice used upon receipt from Charles River company, n = 8 for Swiss Webster mice bred at Georgia State University. Significance was determined using two-way group ANOVA (#P < 0.05 compared to water-treated group). b′–t′, Dietary emulsifiers induce perturbations in faecal bile acids composition. Faecal bile acids composition was analysed at the Metabolomics Core of the University of Michigan. Lithocholic acid (LCA; b′), chenodeoxycholic acid (CDCA; c′), deoxycholic acid (DCA; d′), hyodeoxycholic acid/ursodeoxycholic acid (HDCA/UDCA; e′), α-muricholic acid (α-MCA; f′), β-muricholic acid (β -MCA; g′), cholic acid (CA; h′), hyocholic acid (HCA; i′), ω-muricholic acid (ω-MCA; j′), glycolithocholic acid (GLCA; k′), glycochenodeoxycholic acid (GCDCA; l′), glycodeoxycholic acid (GDCA; m′), hyodeoxycholic acid/glycoursodeoxycholic acid (HDCA/GUDCA; n′), glycocholic acid (GCA; o′), taurolithocholic acid (TLCA; p′), taurine-conjugated chenodeoxycholic acid (TCDCA; q′), taurodeoxycholic acid/tauroursodeoxycholic acid (TDCA/TUDCA; r′), taurohyodeoxycholic acid (s′), and taurocholic acid (TCA; t′) were analysed. Data are the means ± s.e.m. (n = 5, 8 and 7 for water-, CMC- and P80-treated conventional mice groups, respectively; n = 4, 4 and 5 for water-, CMC- and P80-treated germ-free mice groups, respectively). Significance was determined using one-way ANOVA corrected for multiple comparisons with a Sidak test; P < 0.05 compared to water-treated group.

Extended Data Figure 10 Faecal transplants transfer some effects of emulsifers.

a–h, Dietary emulsifiers do not alter mucus thickness under germ-free conditions. a–h, Conventionally-housed (a–c, g–h) and germ-free Swiss Webster (SW; d–f, h) mice were exposed to drinking water containing CMC or P80 (1.0%) for 12 weeks. a–f, Confocal microscopy analysis of microbiota localization: MUC2, green; actin, purple; bacteria, red; and DNA, blue. Scale bar, 20 μm. g, Distances of closest bacteria to intestinal epithelial cells (IEC) per condition over five high-powered fields per mouse. Pictures are representative of five biological replicates. h, Mucus thickness over five high-powered fields per mouse. Data are the means ± s.e.m., n = 5. Significance was determined using one-way ANOVA corrected for multiple comparisons with a Sidak test; P < 0.05 compared to water-treated group. i–l, Dietary emulsifiers do not induce drastic perturbations of mucus layer integrity under germ-free conditions. Germ-free Swiss Webster mice were exposed to drinking water containing CMC or P80 (1.0%) for 8 weeks. Mice were removed from the isolator, inoculated with 0.5 μm green fluorescent beads (Polysciences, Warrington, Pennsylvania), and euthanized 7 h post-inoculation. i–k, Confocal microscopy analysis of fluorescent beads localization: MUC2, green; actin, purple; fluorescent beads, red; and DNA, blue. Scale bar, 20 μm. l, Distances of closest fluorescent beads to intestinal epithelial cells per condition over five high-powered fields per mouse. Pictures are representatives of five biological replicates. Data are the means ± s.e.m., n = 4. Significance was determined using one-way ANOVA corrected for multiple comparisons with a Sidak test. m–r, Dietary emulsifiers increase pro-inflammatory potential of intestinal microbiota in Swiss Webster mice, transferable to germ-free mice recipients. m, n, Germ-free Swiss Webster mice were exposed to drinking water containing CMC or P80 (1.0%) for 12 weeks. Bioactive levels of faecal flagellin (m) and LPS (n) were assayed with TLR5 and TLR4 reporter cells. o, p, Swiss Webster mice were exposed to drinking water containing CMC or P80 (1.0%) for 12 weeks. Bioactive levels of faecal flagellin (o) and LPS (p) were assayed with TLR5 and TLR4 reporter cells. q, r, Germ-free Swiss Webster mice were conventionalized via microbiota transplant from the Swiss Webster mice treated with emulsifiers described above. Bioactive levels of faecal flagellin (q) and LPS (r) were assayed with TLR5 and TLR4 reporter cells. Data are the means ± s.e.m. n = 4 for germ-free mice, n = 8 for conventionally housed mice and n = 5 for conventionalized mice. Significance was determined using two-way ANOVA corrected for multiple comparisons with a Bonferroni test; P < 0.05 compared to control group. s–y, Microbiota transplant transfers emulsifier-induced low-grade intestinal inflammation. Germ-free Swiss Webster mice were conventionalized via microbiota transplant from mice that received standard drinking water or drinking water containing CMC or P80 (1.0%). s, Schematic representation of the experiment. t, Faecal levels of the inflammatory marker LCN2 over time; u, myeloperoxidase levels; v, colon weights; w, colon lengths; x, spleen weights; and y, food intake measurement. Data are the means ± s.e.m., n = 4. Significance was determined using one-way ANOVA corrected for multiple comparisons with a Sidak test or using two-way ANOVA corrected for multiple comparisons with a Bonferroni test; P < 0.05 compared to control group. z–e′, Dietary emulsifiers induce profound alterations in gut microbiota composition in Swiss Webster mice, transferable to germ-free mice recipients. z–b′, Swiss Webster mice were exposed to drinking water containing CMC or P80 (1.0%) for 12 weeks. Principal coordinates analysis (PCoA) of the unweighted UniFrac distance matrix of faecal microbiota at day 0 (z), day 42 (a′) and day 98 (b′). c′–e′, Germ-free Swiss Webster mice were conventionalized via microbiota transplant from the Swiss Webster mice treated with emulsifiers described above. PCoA of the unweighted UniFrac distances of faecal microbiota at day 14 (c′), day 42 (d′) and day 98 (e′) post-transplant. For z–b′, n = 5, 8 and 7 for water-, CMC- and P80-treated groups, respectively; for c′–e′, n = 5, 4 and 3 for water-, CMC- and P80-treated groups, respectively. Treatment of each mouse is indicated by point colour and matching coloured circles indicate mice receiving the same treatment (blue, water; orange, CMC; purple, P80). Black dashed circles represent mice sharing a cage.

Supplementary information

Supplementary Table 1

This table contains analysis of taxonomic abundances at the phyla, class, order and family level. WT, IL10-/- and TLR5-/- mice were exposed to drinking water containing CMC or P80 (1.0%) for 12 weeks. Microbiota composition was analyzed. Taxonomic abundances were analyzed at different levels (phyla, class, order and family). All the significantly altered groups upon emulsifier exposure are highlighted in bold. p-values were calculated using a 2-tailed t-test. (PDF 1944 kb)

Supplementary Table 2

This table shows OTUs statistically different between water-treated group and emulsifier-treated group. WT, IL10-/- and TLR5-/- mice were exposed to drinking water containing CMC or P80 (1.0%) for 12 weeks. Microbiota composition was analyzed. Table lists all OTUs found to be statistically different between water-treated group and emulsifier-treated groups. All OTUs that were previously described to have mucolytic properties are highlighted in purple. p-values were calculated using a 2-tailed t-test. (PDF 689 kb)

Supplementary Table 3

This table contains prevalence analysis of the OTUs found to be related to Helicobacter genus. WT, IL10-/- and TLR5-/- mice were exposed to drinking water containing CMC or P80 (1.0%) for 12 weeks. Microbiota composition was analyzed. Prevalence of the OTUs 470487, 2729098, 102480 and 3319464 (Greengenes Prok_MSA IDs), assigned to belong to the Helicobacter genus, were analyzed. p-values were calculated using a 2-tailed t-test. (PDF 88 kb)

PowerPoint slides

Rights and permissions

About this article

Cite this article

Chassaing, B., Koren, O., Goodrich, J. et al. Dietary emulsifiers impact the mouse gut microbiota promoting colitis and metabolic syndrome.Nature 519, 92–96 (2015). https://doi.org/10.1038/nature14232

Download citation