The composition of the gut microbiota shapes the colon mucus barrier

In the paper

“The thickness and growth of the colon mucus were similar in the two colonies.”

• Also stated in: § Differences in the mucus quality in mice from the two husbandries · plain

  “The thickness of the inner mucus layer as well as the mucus growth did not differ significantly between the two rooms on either food (Fig3B).”

In the paper

“Immunohistochemistry revealed that the bacteria were separated from the epithelium by a well-stratified mucus layer in Room 1, but bacteria were in close proximity to the epithelium of mice colonized with microbiota from Room 2 (Fig5D).”

In the paper

“On Food A, the mice bred in Room 1 were significantly heavier compared to mice bred in Room 2 (P = 0.022, Supplementary Fig S1A).”

In the paper

“The small intestinal mucus is normally easily removed by aspiration, and the thickness was measured before (pre) and after (post) aspiration. No differences were observed between the rooms or diets (Fig3A).”

In the paper

“The major difference between the lumen- and mucus-associated microbiota was found in the distal colon where the phyla Deferribacteres had higher abundance in both rooms (Supplementary Fig S4, Table1).”

In the paper

“When bacteria were localized in relation to the colon mucus using fluorescent in situ hybridization, bacteria penetrated the inner mucus layer at distinct locations. A separation between epithelium and bacteria was observed in other areas and most bacteria were still not in direct contact with the epithelial surface.”

In the paper

“Within this phylum, the classes Betaproteobacteria and Epsilonproteobacteria were found at higher levels in the mucus of mice in Room 2 and Epsilonproteobacteria were significantly more abundant in distal colon lumen and caecum (Fig1D, Table1).”

In the paper

“The dominant genus within this class contributing to this difference was the genus Allobaculum, which was also found in higher relative abundances at all locations and significantly so in the lumen samples (Fig2B, Table1).”

In the paper

“Altering the diet from weaning gave some shift in the microbiota, but less than the difference between the two rooms.”

In the paper

“A tendency towards longer crypts in mice from Room 2 was also observed, but this did not reach significance (Fig4D).”

In the paper

“The inner mucus layer was after fixation observed to be thinner, and stratification was not evident in sections from animals fed Food B in both rooms, but the difference between the two rooms remained (Fig4B).”

In the paper

“The stool of mice fed Food A was hard and formed distinct pellets, while the stool in animals fed Food B had a looser consistency and did not form hard pellets.”

In the paper

“When bacterial diversity was studied (Supplementary Figs S2 and S3), the ileum showed the lowest diversity and the distal colon the highest.”

In the paper

“The labelled cells were significantly increased in mice from Room 2 as compared to Room 1 (Fig4F and H).”

In the paper

“Principal Coordinates Unweighted UniFrac analysis 20 showed separation of ileum and colon samples along the first component, whereas the two rooms clearly separated along PC2 on Food A (Fig1A and B).”

What the sources say

• Not checked yetcites [20] Lozupone C, Knight R (2005) UniFrac: a new phylogenetic method for comparing microbial communities. Appl Environ Microbiol 71: 8228–8235 source

In the paper

“Inflammatory markers as S100a8, S100a9 and eosinophil cationic protein 3 were not detected and no difference in albumin or alpha 2-macroglobulin was found between the groups.”

In the paper

“As shown before, mice from Room 1 secreted mucus that was impenetrable to the beads, whereas the mucus on explants from mice housed in Room 2 was more penetrable (Fig3C).”

In the paper

“Mucus penetrability phenotypes were again different as a more penetrable inner mucus layer was observed in mice colonized with microbiota from Room 2, while mice colonized with microbiota from Room 1 showed a mucus layer impervious to beads (Fig5B and C).”

In the paper

“All mice on Food B showed higher relative abundances of the phylum Proteobacteria at all locations along the gut and in both rooms with significant differences in ileum lumen, ileum mucus and caecum in Room 1, and in distal colon lumen in both rooms (Supplementary Fig S5, Supplementary Table S1).”

In the paper

“Mice given food B showed increased penetrability in both groups, but a difference still remained between Room 1 and 2 mice (Fig3C).”

In the paper

“To analyse the reasons for the different mucus properties, proteomic analyses were performed showing no major differences between the rooms and a high correlation of the proteins identified in the two rooms (Supplementary Fig S6D and E).”

In the paper

“In ileum mucus, the dominant genus within the Clostridia class was Candidatus arthromitus (segmented filamentous bacteria, SFB), which was also found in higher levels in Room 2 (Table1). SFB also showed a small but significant difference between the rooms in the distal colon with higher levels in Room 2.”

In the paper

“The thickness of the inner mucus layer as well as the mucus growth did not differ significantly between the two rooms on either food (Fig3B).”

In the paper

“The number of these cells was higher in the Room 2 animals; however, it did not reach significance (Fig4G and Supplementary Fig S7A), but there was no major difference in the overall histology between the groups (Supplementary Fig S7B and C).”

In the paper

“Our study corroborates previous studies showing an association between altered diets and an increase in Erysipelotrichi and Allobaculum, but various effects are shown for fat-enriched diets 29, 30, 31, 32.”

What the sources say

• Not checked yetcites [29] Everard A, Lazarevic V, Gaia N, Johansson M, Stahlman M, Backhed F, Delzenne NM, Schrenzel J, Francois P, Cani PD (2014) Microbiome of prebiotic‐treated mice reveals novel targets involved in host response during obesity. ISME J 8: 2116–2130 source
• Not checked yetcites [30] Islam KB, Fukiya S, Hagio M, Fujii N, Ishizuka S, Ooka T, Ogura Y, Hayashi T, Yokota A (2011) Bile acid is a host factor that regulates the composition of the cecal microbiota in rats. Gastroenterology 141: 1773–1781 source
• Not checked yetcites [31] Martinez I, Wallace G, Zhang C, Legge R, Benson AK, Carr TP, Moriyama EN, Walter J (2009) Diet‐induced metabolic improvements in a hamster model of hypercholesterolemia are strongly linked to alterations of the gut microbiota. Appl Environ Microbiol 75: 4175–4184 source
• Not checked yetcites [32] Turnbaugh PJ, Ridaura VK, Faith JJ, Rey FE, Knight R, Gordon JI (2009) The effect of diet on the human gut microbiome: a metagenomic analysis in humanized gnotobiotic mice. Sci Transl Med 1: 6ra14 source

In the paper

“We have also observed different inner mucus phenotypes in animals from different vendors, further emphasizing the need for standardization and caution.”

In the paper

“Even though the present study reveals subtle differences in the microbiota between the two mouse colonies, an increased inflammatory tone was observed in Room 2.”

In the paper

“Muc2-deficient mice totally lack a protective inner mucus layer and have direct contact of bacteria with the epithelium and these mice develop severe colitis 14.”

What the sources say

• Not checked yetcites [14] Johansson ME, Phillipson M, Petersson J, Velcich A, Holm L, Hansson GC (2008) The inner of the two Muc2 mucin‐dependent mucus layers in colon is devoid of bacteria. Proc Natl Acad Sci USA 105: 15064–15069 source

In the paper

“The different properties of the mucus depended on the microbiota, as they were transmissible by transfer of caecal microbiota to germ-free mice.”

• Also stated in: § The two different mucus phenotypes were reproduced by colonization of germ-free mice · plain

  “The composition of intestinal microbiota thus has a major influence on the properties of the inner colon mucus barrier.”

In the paper

“Thus, our study shows that bacteria and their community structure affect mucus barrier properties in ways that can have implications for health and disease.”

In the paper

“The mucus is structurally built around the MUC2 mucin that after secretion from the goblet cell expands dramatically and forms sheets of MUC2 networks that stack and form the stratified mucus layer 16.”

What the sources say

• Not checked yetcites [16] Ambort D, Johansson ME, Gustafsson JK, Nilsson HE, Ermund A, Johansson BR, Koeck PJ, Hebert H, Hansson GC (2012) Calcium and pH‐dependent packing and release of the gel‐forming MUC2 mucin. Proc Natl Acad Sci USA 109: 5645–5650 source

In the paper

“Deficiencies in decorating the mucus with O-glycans cause a faster degradation of the Muc2 mucin network resulting in increased bacterial contact with the cell surface and development of spontaneous colitis 19.”

What the sources say

• Not checked yetcites [19] Fu J, Wei B, Wen T, Johansson ME, Liu X, Bradford E, Thomsson KA, McGee S, Mansour L, Tong M et al (2011) Loss of intestinal core 1‐derived O‐glycans causes spontaneous colitis in mice. J Clin Invest 121: 1657–1666 source

In the paper

“It was thus concluded that the two C57BL/6 mice strains bred separately had stable, but different microbiota that were vertically transmitted.”

In the paper

“Nevertheless, these mice often had an even more developed mucus layer than observed in Room 1, which further supports the influence of the microbiota on mucus and that the Room 1 can be considered as a reasonable model.”

In the paper

“The two colonies were maintained on different diets with different composition, but we controlled for the impact of diet, at least within one generation of offspring, by feeding the mice in the two colonies the same chow (Food A, standard chow or Food B, autoclaved chow) from weaning.”

In the paper

“The exoglycosidases are usually arranged in specialized genetic loci for utilizing specific types of substrates as, for example, starch (Sus) and xylose-containing polysaccharides (PUL) 27.”

What the sources say

• Not checked yetcites [27] Larsbrink J, Rogers TE, Hemsworth GR, McKee LS, Tauzin AS, Spadiut O, Klinter S, Pudlo NA, Urs K, Koropatkin NM et al (2014) A discrete genetic locus confers xyloglucan metabolism in select human gut Bacteroidetes. Nature 506: 498–502 source

In the paper

“Thus, free-living mice have mucus that is even better developed, thicker and more stratified with bacteria further separated from the epithelium than in Room 1.”

In the paper

“The commensal bacteria live in the non-attached and expanded outer mucus of colon 26, where they can cleave off and utilize mucin glycans as energy source.”

What the sources say

• Not checked yetcites [26] Johansson ME, Holmen Larsson JM, Hansson GC (2010) Microbes and Health Sackler Colloquium: the two mucus layers of colon are organized by the MUC2 mucin, whereas the outer layer is a legislator of host‐microbial interactions. Proc Natl Acad Sci USA 108: 4659–4665 source

In the paper

“The protein differences were small and not likely to explain the variable mucus properties.”

In the paper

“Ulcerative colitis (UC) patients with active disease have bacteria in contact with the epithelium, whereas patients with UC in remission show a more mixed picture 15.”

What the sources say

• Not checked yetcites [15] Johansson ME, Gustafsson JK, Holmen‐Larsson J, Jabbar KS, Xia L, Xu H, Ghishan FK, Carvalho FA, Gewirtz AT, Sjovall H et al (2014) Bacteria penetrate the normally impenetrable inner colon mucus layer in both murine colitis models and patients with ulcerative colitis. Gut 63: 281–291 source

In the paper

“The origin of the germ-free mice is more related to the origin of the animals in Room 2, but as they still were able to form a functional mucus barrier upon colonization with flora from animals in Room 1, genetic influences are less likely.”

In the paper

“TM7 and the genus Prevotella are also increased in mice deficient in the inflammasome component Nlrp6 and suggested to be associated with inflammation 51.”

What the sources say

• Not checked yetcites [51] Hu B, Elinav E, Huber S, Strowig T, Hao L, Hafemann A, Jin C, Wunderlich C, Wunderlich T, Eisenbarth SC et al (2013) Microbiota‐induced activation of epithelial IL‐6 signaling links inflammasome‐driven inflammation with transmissible cancer. Proc Natl Acad Sci USA 110: 9862–9867 source

In the paper

“However, it is considered to foster a normal intestinal homeostasis in healthy mice, something that is in contrast to the here observed less developed colon mucus 52.”

What the sources say

• Not checked yetcites [52] Schnupf P, Gaboriau‐Routhiau V, Cerf‐Bensussan N (2013) Host interactions with Segmented Filamentous Bacteria: an unusual trade‐off that drives the post‐natal maturation of the gut immune system. Semin Immunol 25: 342–351 source

In the paper

“Certain bacteria can, in contrast to promoting well-developed mucus, also destroy the mucus and its protective properties.”

What the sources say

• Not checked yetcites [36] Bergstrom KS, Kissoon‐Singh V, Gibson DL, Ma C, Montero M, Sham HP, Ryz N, Huang T, Velcich A, Finlay BB et al (2010) Muc2 protects against lethal infectious colitis by disassociating pathogenic and commensal bacteria from the colonic mucosa. PLoS Pathog 6: e1000902 source
• Not checked yetcites [37] van der Post S, Subramani DB, Backstrom M, Johansson ME, Vester‐Christensen MB, Mandel U, Bennett EP, Clausen H, Dahlen G, Sroka A et al (2013) Site‐specific O‐glycosylation on the MUC2 mucin protein inhibits cleavage by the Porphyromonas gingivalis secreted cysteine protease (RgpB). J Biol Chem 288: 14636–14646 source
• Not checked yetcites [38] Zarepour M, Bhullar K, Montero M, Ma C, Huang T, Velcich A, Xia L, Vallance BA (2013) The mucin Muc2 limits pathogen burdens and epithelial barrier dysfunction during Salmonella enterica serovar Typhimurium colitis. Infect Immun 81: 3672–3683 source

In the paper

“The TM7 phylum is also found in human stool samples at low levels 47 and proposed to play a role in intestinal inflammation 48.”

What the sources say

• Not checked yetcites [47] Segata N, Haake SK, Mannon P, Lemon KP, Waldron L, Gevers D, Huttenhower C, Izard J (2012) Composition of the adult digestive tract bacterial microbiome based on seven mouth surfaces, tonsils, throat and stool samples. Genome Biol 13: R42 source
• Not checked yetcites [48] Kuehbacher T, Rehman A, Lepage P, Hellmig S, Folsch UR, Schreiber S, Ott SJ (2008) Intestinal TM7 bacterial phylogenies in active inflammatory bowel disease. J Med Microbiol 57: 1569–1576 source

In the paper

“The class Deltaproteobacteria contains sulphur-reducing bacteria (SRB) that are also found at higher frequencies in patients with IBD as compared to healthy individuals 41.”

What the sources say

• Not checked yetcites [41] Verma R, Verma AK, Ahuja V, Paul J (2010) Real‐time analysis of mucosal flora in patients with inflammatory bowel disease in India. J Clin Microbiol 48: 4279–4282 source

In the paper

“Mouse colitis models typically show features like crypt elongation due to increased cell proliferation, infiltration of immune cells in lamina propria, thickening of muscularis propria and weight loss, all criteria used for scoring colitis 33, 34.”

What the sources say

• Not checked yetcites [33] Sellon RK, Tonkonogy S, Schultz M, Dieleman LA, Grenther W, Balish E, Rennick DM, Sartor RB (1998) Resident enteric bacteria are necessary for development of spontaneous colitis and immune system activation in interleukin‐10‐deficient mice. Infect Immun 66: 5224–5231 source
• Not checked yetcites [34] Van der SM, de Koning BA, De Bruijn AC, Velcich A, Meijerink JP, Van Goudoever JB, Buller HA, Dekker J, Van Seuningen I, Renes IB et al (2006) Muc2‐deficient mice spontaneously develop colitis, indicating that MUC2 is critical for colonic protection. Gastroenterology 131: 117–129 source

In the paper

“This phylum has been linked to inflammatory conditions of the mouth such as periodontitis 46.”

What the sources say

• Not checked yetcites [46] Liu B, Faller LL, Klitgord N, Mazumdar V, Ghodsi M, Sommer DD, Gibbons TR, Treangen TJ, Chang YC, Li S et al (2012) Deep sequencing of the oral microbiome reveals signatures of periodontal disease. PLoS ONE 7: e37919 source

In the paper

“This is a phylum, with no cultivated representatives, commonly found in the oral flora at low levels 44, 45.”

What the sources say

• Not checked yetcites [44] Brinig MM, Lepp PW, Ouverney CC, Armitage GC, Relman DA (2003) Prevalence of bacteria of division TM7 in human subgingival plaque and their association with disease. Appl Environ Microbiol 69: 1687–1694 source
• Not checked yetcites [45] Podar M, Abulencia CB, Walcher M, Hutchison D, Zengler K, Garcia JA, Holland T, Cotton D, Hauser L, Keller M (2007) Targeted access to the genomes of low‐abundance organisms in complex microbial communities. Appl Environ Microbiol 73: 3205–3214 source

In the paper

“For the microbiota analysis, significant differences were conducted using the Wilcoxon rank-sum test, and P-values were converted to false discovery rate values (Q-values) to correct for multiple testing in the R software (http://www.r-project.org/).”

In the paper

“The TM7 phylum has also been found in mice more susceptible to colitis caused by Helicobacter hepaticus 49.”

What the sources say

• Not checked yetcites [49] Yang I, Eibach D, Kops F, Brenneke B, Woltemate S, Schulze J, Bleich A, Gruber AD, Muthupalani S, Fox JG et al (2013) Intestinal microbiota composition of interleukin‐10 deficient C57BL/6J mice and susceptibility to Helicobacter hepaticus‐induced colitis. PLoS ONE 8: e70783 source

In the paper

“The phylum Proteobacteria has been associated with human Crohn's disease 40.”

What the sources say

• Not checked yetcites [40] Gophna U, Sommerfeld K, Gophna S, Doolittle WF, Veldhuyzen van Zanten SJ (2006) Differences between tissue‐associated intestinal microfloras of patients with Crohn's disease and ulcerative colitis. J Clin Microbiol 44: 4136–4141 source

In the paper

“Species of lower taxonomic levels within the Proteobacteria phylum have been correlated with colitis in a maternally transmitted fashion 39.”

What the sources say

• Not checked yetcites [39] Garrett WS, Gallini CA, Yatsunenko T, Michaud M, DuBois A, Delaney ML, Punit S, Karlsson M, Bry L, Glickman JN et al (2010) Enterobacteriaceae act in concert with the gut microbiota to induce spontaneous and maternally transmitted colitis. Cell Host Microbe 8: 292–300 source

In the paper

“It is obvious from this study that the microbiota influences the properties of the mucus barrier and that this can be observed within the same animal house with animals of identical genetic background.”

In the paper

“The microbiota mucus sample strategy used may have reduced the observed differences in mucus microbiota as the amounts of attached mucus were not controlled for.”

In the paper

“In especially the core 1 deficient mice, the inner mucus layer was more vulnerable and quickly dissolved allowing bacteria to reach the epithelium and by this trigger inflammation 19.”

What the sources say

• Not checked yetcites [19] Fu J, Wei B, Wen T, Johansson ME, Liu X, Bradford E, Thomsson KA, McGee S, Mansour L, Tong M et al (2011) Loss of intestinal core 1‐derived O‐glycans causes spontaneous colitis in mice. J Clin Invest 121: 1657–1666 source