Dietary emulsifiers impact the mouse gut microbiota promoting colitis and metabolic syndrome

  • A Corrigendum to this article was published on 04 May 2016

Abstract

The intestinal tract is inhabited by a large and diverse community of microbes collectively referred to as the gut microbiota. While the gut microbiota provides important benefits to its host, especially in metabolism and immune development, disturbance of the microbiota–host relationship is associated with numerous chronic inflammatory diseases, including inflammatory bowel disease and the group of obesity-associated diseases collectively referred to as metabolic syndrome. A primary means by which the intestine is protected from its microbiota is via multi-layered mucus structures that cover the intestinal surface, thereby allowing the vast majority of gut bacteria to be kept at a safe distance from epithelial cells that line the intestine1. Thus, agents that disrupt mucus–bacterial interactions might have the potential to promote diseases associated with gut inflammation. Consequently, it has been hypothesized that emulsifiers, detergent-like molecules that are a ubiquitous component of processed foods and that can increase bacterial translocation across epithelia in vitro2, might be promoting the increase in inflammatory bowel disease observed since the mid-twentieth century3. Here we report that, in mice, relatively low concentrations of two commonly used emulsifiers, namely carboxymethylcellulose and polysorbate-80, induced low-grade inflammation and obesity/metabolic syndrome in wild-type hosts and promoted robust colitis in mice predisposed to this disorder. Emulsifier-induced metabolic syndrome was associated with microbiota encroachment, altered species composition and increased pro-inflammatory potential. Use of germ-free mice and faecal transplants indicated that such changes in microbiota were necessary and sufficient for both low-grade inflammation and metabolic syndrome. These results support the emerging concept that perturbed host–microbiota interactions resulting in low-grade inflammation can promote adiposity and its associated metabolic effects. Moreover, they suggest that the broad use of emulsifying agents might be contributing to an increased societal incidence of obesity/metabolic syndrome and other chronic inflammatory diseases.

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Figure 1: Dietary emulsifiers alter microbiota localization, composition, and pro-inflammatory potential.
Figure 2: Dietary emulsifiers promote colitis in susceptible mice and low-grade intestinal inflammation in wild-type mice.
Figure 3: Dietary emulsifiers promote metabolic syndrome.
Figure 4: Altered microbiota is necessary and sufficient for emulsifier-induced metabolic syndrome.

Accession codes

Primary accessions

European Nucleotide Archive

Data deposits

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

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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

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Authors

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 to Andrew T. Gewirtz.

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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. ac, 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. ej, 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. eg, 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. ko, 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). mo, 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. pt, 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. ad, 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. ad, 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. hn, 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. hk, 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. ad, 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. eh, As for ad with Il10−/− mice. il, As for ad 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. ag, 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. sv, 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. dh, 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 eg, 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. ik, 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 R2 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. ag, 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. hk, 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. lt, 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 (ua′) 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). ls, 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. tw, 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). xf′, 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. ac, 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. gp, Emulsifier-induced low-grade intestinal inflammation was abolished under germ-free conditions. Conventionally housed (gk) and germ-free (lp) 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. qw, 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. xa′, 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. ah, Conventionally-housed (ac, gh) and germ-free Swiss Webster (SW; df, h) mice were exposed to drinking water containing CMC or P80 (1.0%) for 12 weeks. af, 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. il, 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. ik, 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. mr, 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. sy, 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. ze′, Dietary emulsifiers induce profound alterations in gut microbiota composition in Swiss Webster mice, transferable to germ-free mice recipients. zb′, 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 zb′, 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.

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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)

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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

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