{"@context":"https://schema.org","@type":"Article","headline":"The composition of the gut microbiota shapes the colon mu...","description":"Scientific Report18 December 2014Open Access Source Data The composition of the gut microbiota shapes the colon mucus barrier Hedvig E Jakobsson Hedvig E Jakobsson Department of Medical Biochemistry, University of Gothenburg, Gothenburg, Sweden Search for more papers by this author Ana M Rodríguez-Piñeiro Ana M Rodríguez-Piñeiro Department of Medical Biochemistry, University of Gothenburg, Gothenburg, Sweden Search for more papers by this author André Schütte André Schütte Department of Medical Biochemistry, University of Gothenburg, Gothenburg, Sweden Search for more papers by this author Anna Ermund Anna Ermund Department of Medical Biochemistry, University of Gothenburg, Gothenburg, Sweden Search for more papers by this author Preben Boysen Preben Boysen Department of Food Safety and Infection Biology, Faculty of Veterinary Medicine and Biosciences, Norwegian University of Life Sciences, Oslo, Norway Search for more papers by this author Mats Bemark Mats Bemark Department of Microbiology and Immunology, University of Gothenburg, Gothenburg, Sweden Department of Clinical Immunology and Transfusion Medicine, Sahlgrenska University Hospital, Gothenburg, Sweden Search for more papers by this author Felix Sommer Felix Sommer The Wallenberg Laboratory, Department of Molecular and Clinical Medicine, University of Gothenburg, Gothenburg, Sweden Search for more papers by this author Fredrik Bäckhed Fredrik Bäckhed The Wallenberg Laboratory, Department of Molecular and Clinical Medicine, University of Gothenburg, Gothenburg, Sweden Search for more papers by this author Gunnar C Hansson Gunnar C Hansson Department of Medical Biochemistry, University of Gothenburg, Gothenburg, Sweden Search for more papers by this author Malin EV Johansson Corresponding Author Malin EV Johansson Department of Medical Biochemistry, University of Gothenburg, Gothenburg, Sweden Search for more papers by this author Hedvig E Jakobsson Hedvig E Jakobsson Department of Medical Biochemistry, University of Gothenburg, Gothenburg, Sweden Search for more papers by this author Ana M Rodríguez-Piñeiro Ana M Rodríguez-Piñeiro Department of Medical Biochemistry, University of Gothenburg, Gothenburg, Sweden Search for more papers by this author André Schütte André Schütte Department of Medical Biochemistry, University of Gothenburg, Gothenburg, Sweden Search for more papers by this author Anna Ermund Anna Ermund Department of Medical Biochemistry, University of Gothenburg, Gothenburg, Sweden Search for more papers by this author Preben Boysen Preben Boysen Department of Food Safety and Infection Biology, Faculty of Veterinary Medicine and Biosciences, Norwegian University of Life Sciences, Oslo, Norway Search for more papers by this author Mats Bemark Mats Bemark Department of Microbiology and Immunology, University of Gothenburg, Gothenburg, Sweden Department of Clinical Immunology and Transfusion Medicine, Sahlgrenska University Hospital, Gothenburg, Sweden Search for more papers by this author Felix Sommer Felix Sommer The Wallenberg Laboratory, Department of Molecular and Clinical Medicine, University of Gothenburg, Gothenburg, Sweden Search for more papers by this author Fredrik Bäckhed Fredrik Bäckhed The Wallenberg Laboratory, Department of Molecular and Clinical Medicine, University of Gothenburg, Gothenburg, Sweden Search for more papers by this author Gunnar C Hansson Gunnar C Hansson Department of Medical Biochemistry, University of Gothenburg, Gothenburg, Sweden Search for more papers by this author Malin EV Johansson Corresponding Author Malin EV Johansson Department of Medical Biochemistry, University of Gothenburg, Gothenburg, Sweden Search for more papers by this author Author Information Hedvig E Jakobsson1, Ana M Rodríguez-Piñeiro1, André Schütte1, Anna Ermund1, Preben Boysen2, Mats Bemark3,4, Felix Sommer5, Fredrik Bäckhed5, Gunnar C Hansson1 and Malin EV Johansson 1 1Department of Medical Biochemistry, University of Gothenburg, Gothenburg, Sweden 2Department of Food Safety and Infection Biology, Faculty of Veterinary Medicine and Biosciences, Norwegian University of Life Sciences, Oslo, Norway 3Department of Microbiology and Immunology, University of Gothenburg, Gothenburg, Sweden 4Department of Clinical Immunology and Transfusion Medicine, Sahlgrenska University Hospital, Gothenburg, Sweden 5The Wallenberg Laboratory, Department of Molecular and Clinical Medicine, University of Gothenburg, Gothenburg, Sweden *Corresponding author. Tel: +46 31 786 3487; E-mail: [email protected] EMBO Reports (2015)16:164-177https://doi.org/10.15252/embr.201439263 See also: H Li et al (February 2015) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Two C57BL/6 mice colonies maintained in two rooms of the same specific pathogen-free (SPF) facility were found to have different gut microbiota and a mucus phenotype that was specific for each colony. The thickness and growth of the colon mucus were similar in the two colonies. However, one colony had mucus that was impenetrable to bacteria or beads the size of bacteria—which is comparable to what we observed in free-living wild mice—whereas the other colony had an inner mucus layer penetrable to bacteria and beads. The different properties of the mucus depended on the microbiota, as they were transmissible by transfer of caecal microbiota to germ-free mice. Mice with an impenetrable mucus layer had increased amounts of Erysipelotrichi, whereas mice with a penetrable mucus layer had higher levels of Proteobacteria and TM7 bacteria in the distal colon mucus. Thus, our study shows that bacteria and their community structure affect mucus barrier properties in ways that can have implications for health and disease. It also highlights that genetically identical animals housed in the same facility can have rather distinct microbiotas and barrier structures. Synopsis Mice of the same strain living in the same animal facility can have distinct microbiotas, which affects the penetrability of the colon mucus barrier in a transmissible manner. Free-living mice have impenetrable mucus, as one of the experimental groups. The colon microbiota influences the penetrability of the inner mucus layer. Certain bacteria are associated with a more penetrable inner mucus layer. The mucus phenotype was transmissible by transfer of the microbiota to germ-free mice. Introduction The distal small intestine and the large intestine are the reservoirs for an enormous and complex community of micro-organisms, the gut microbiota. The density of intestinal microbes forms a gradient along the intestine with few bacteria in the upper small intestine and up to 1012 bacteria per gram of faeces in distal colon 1. The microbiota is diverse and typically made up of in total 500–1,000 species with at least 160 species that are shared among individuals with two phyla, the Bacteroidetes and Firmicutes being dominant 23. Less abundant phyla are Proteobacteria, Verrucomicrobia, Actinobacteria, Fusobacteria and Cyanobacteria. We have co-evolved with our microbiota and developed a finely tuned co-existence, with mutual benefits and ways to avoid harmful effects on the host. The microbiota, for example, aids in food digestion and development of the immune system 4. Together with the mucus layers, a stable microbiota prevents pathogenic bacteria from establishing host infections 56. Mucus covers the intestinal epithelium, but the principle of this mucus protection is solved differently along the intestinal tract 7. The mucus in the small intestine fills up the space between the villi and covers these, but is not attached to the epithelium and has a structure that can allow particles as large as bacteria to penetrate 8. The mucus protection in this site acts as a diffusion barrier with a high concentration of antibacterial products close to the epithelium and few bacteria reaching near the cell surface 910. The small intestinal mucus not only excludes the microbiota, but is also important for immune system development 111213. The higher bacterial load in colon and the slow transit time requires a different protective strategy. Here, mucus forms a physical barrier separating bacteria from the host, which is especially important and developed in the distal colon. The secreted mucus in distal colon forms a stratified inner layer of mucus that is able to keep the bacteria at a distance (> 50 μm in mice, > 200 μm in human) from the epithelium 1415. 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. Failure of this protective barrier to separate bacteria from the epithelium is observed in a number of murine colitis models 1517. 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. Mice with mutations in the Muc2 gene also spontaneously develop inflammation 18. 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. The mucus protection system observed in mouse is also present in human colon where an even thicker inner mucus layer separates bacteria from the tissue 15. 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. The possibility that the microbiota stimulates the host to develop a mucus layer with improved protective properties has not been explored. We have studied the colon mucus barrier in different wild-type C57BL/6 mouse colonies housed and bred separately in the same specific pathogen-free (SPF) mouse facility. The mucus phenotypes were compared with wild normally free-living mice caught in their natural habitat to elucidate the protective properties of natural occurring mucus. The bacterial composition in the two mouse colonies was analysed by 16S rRNA gene sequencing, which revealed specific bacterial compositions in the two colonies. Bacterial transmission of the mucus phenotypes was shown by colonizing germ-free animals with the flora from the two colonies. Results Bacterial composition in the two husbandries In the same SPF animal facility, two different C57BL/6 colonies have been bred in different rooms. These two C57BL/6 colonies were named as Room 1 and Room 2, where our previous results on mucus properties have been based on the strain in Room 1, which was derived from Taconic 14. 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. 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). 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. The microbiota composition of mice in the two rooms was analysed by 16S rRNA gene sequencing of bacteria in the lumen of ileum, distal colon (called lumen) and caecum as well as bound to the mucus on the tissue after washing the ileum and distal colon (called mucus). The sequencing data have been deposited to the European Nucleotide Archive (ENA) with accession number PRJEB7982. The mucus bacteria thus reflected the bacteria either attached to the outer border of the inner colon mucus layer, within the inner colon mucus in colon, or attached to the epithelial cells of the small intestine. Analysing the data using QIIME revealed that the microbiota differed between Room 1 and Room 2. 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 (Fig 1A and B). The difference remained also when mice were given Food B (Supplementary Fig S1). When bacterial diversity was studied (Supplementary Figs S2 and S3), the ileum showed the lowest diversity and the distal colon the highest. The microbiota throughout the gut thus clearly differed between the two colonies housed in adjacent rooms in the same animal facility. Figure 1. Microbiota composition in the two mouse colonies Unweighted UniFrac-based PCoA plot of gut microbial communities from mice in Room 1 (red) and Room 2 (blue) at all intestinal locations. Unweighted UniFrac-based PCoA plot of mice gut microbial communities from both rooms at different gut segments; ileum lumen (blue), ileum mucus (yellow), distal colon lumen (red), distal colon mucus (green) and caecum (brown) (data as in A). The relative abundance (%) of the phylum TM7 in ileum lumen Room 1 (n = 11) and Room 2 (n = 11), ileum mucus Room 1 (n = 11) and Room 2 (n = 11), distal colon lumen Room 1 (n = 11) and Room 2 (n = 11), distal colon mucus Room 1 (n = 9) and Room 2 (n = 7), and caecum Room 1 (n = 11) and Room 2 (n = 11). The relative abundance (%) of the phylum Proteobacteria (beta and epsilon) in distal colon lumen and mucus in Room 1 (n = 11 in lumen and n = 9 in mucus) and Room 2 (n = 11 in lumen and n = 7 in mucus). Class level microbiota composition in mice from Room 1 and Room 2. The mean relative abundance (%) of the most abundant bacterial classes in ileum lumen Room 1 (n = 11) and Room 2 (n = 11), ileum mucus Room 1 (n = 11) and Room 2 (n = 11), distal colon lumen Room 1 (n = 11) and Room 2 (n = 11), distal colon mucus Room 1 (n = 9) and Room 2 (n = 7), and caecum Room 1 (n = 11) and Room 2 (n = 11). Classes represented as less than 1%: Other, DA052, Actinobacteria, Coriobacteriia, Cytophagia, Flavobacteriia, Sphingobacteriia, (Saprospirae), 4C0d-2, Chloroplast, Deferribacteres, ZB2, Alphaproteobacteria, Betaproteobacteria, Epsilonproteobacteria, Deltaproteobacteria, Gammaproteobacteria, TM7-3, Mollicutes, Verrucomicrobiae. Data information: In (C) and (D), data are presented as mean ± SD. Wilcoxon's rank-sum test was conducted to compare over- or under-representation of the phylum. To correct for multiple testing, the P-values were converted to false discovery rate values (Q-values). *P < 0.001, Q < 0.001, **P < 0.001, Q < 0.01. Source data are available online for this figure. Source Data for Figure 1 [embr201439263-SourceData-Fig1.xlsx] Download figure Download PowerPoint The different microbiota was reflected in significant differences at multiple taxonomical levels (Table 1). However, the relative amounts of the dominant phyla (Firmicutes and Bacteroidetes) did not significantly differ between the two rooms at any location throughout the gut (Supplementary Fig S4, Table 1). 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, Table 1). Table 1. The mean of the relative abundance of dominant phyla, classes and genera in ileum, distal colon, and caecum samples obtained from mice bred in Room 1 or Room 2 Ileum lumen Ileum mucus Distal colon lumen Distal colon mucus Caecum Room 1 Room 2 Room 1 Room 2 Room 1 Room 2 Room 1 Room 2 Room 1 Room 2 n = 11 n = 11 n = 11 n = 11 n = 11 n = 11 n = 9 n = 7 n = 11 n = 11 mean % (SD) mean % (SD) mean % (SD) mean % (SD) mean % (SD) mean % (SD) mean % (SD) mean % (SD) mean % (SD) mean % (SD) Firmicutes 88 (6) 92 (6) 81 (9) 88 (9) 80 (10) 71 (8) 73 (11) 62 (24) 85 (3) 83 (3) Bacilli 50 (19) 76 (15) 50 (23) 38 (24) 28 (12) 34 (19) 15 (11) 17 (14) 12 (5) 7 (4) Lactobacillus 50 (19) 75 (15) 50 (21) 38 (24) 27 (12) 34 (19) 15 (11) 16 (9) 12 (5) 7 (4) Clostridia 2 (1) 11 (11) 15 (23)** 47 (28)** 30 (20) 35 (22) 50 (22) 45 (28) 64 (11)** 75 (5)** Unknown genus (family unknown) < 1 < 1 1 (2) < 1 10 (9) 12 (14) 18 (10) 16 (17) 27 (13) 29 (19) Other < 1 < 1 2 (4) < 1 10 (8) 9 (6) 18 (11) 13 (14) 21 (7) 19 (16) Ruminococcus 0.1 (0.01)*** 0.3 (0.2)*** < 1 < 1 < 1 < 1 < 1 < 1 0.7 (0.3)*** 1 (1)*** Candidatus arthromitus (Segmented filamentous bacteria) 0.04 (0.12)*** 1 (2)*** 11 (23) 43 (29) 0.01 (0.02)*** 0.2 (0.3)*** 0.03 (0.1)*** 1 (1)*** < 1 < 1 Anaerostipes 0.06 (0.05)** –** 0.2 (0.2)** –** 2 (2)** < 1** 0.8 (0.8)*** –*** 0.8 (0.6)** –** Dehalobacterium < 1 < 1 < 1 < 1 < 1 < 1 < 1 < 1 0.2 (0.1)** 0.6 (0.2)** Erysipelotrichi 36 (16)* 5 (6)* 17 (13)** 3 (3)** 22 (14)* 2 (2)* 8 (7) 1 (1) 9 (7)* 0.6 (0.7)* Allobaculum 35 (17)** 5 (6)** 15 (13) 3 (3) 22 (14)** 2 (2)** 7 (7) 1 (1) 9 (7)** 0.4 (0.4)** Other 1 (1)** –** < 1 < 1 0.5 (0.8)*** 0.003 (0.01)*** 0.2 (0.3)*** –*** 0.2 (0.2)** –** Bacteroidetes 11 (6) 6 (5) 18 (9) 10 (8) 19 (10) 27 (8) 12 (6) 17 (14) 13 (4) 13 (3) Bacteroidia 11 (5) 6 (5) 18 (9) 10 (8) 19 (10) 27 (8) 12 (6) 17 (14) 13 (4) 13 (3) Unknown genus (family S24-7) 11 (6)** 6 (5)** 16 (10) 10 (8) 15 (9) 14 (6) 9 (5) 10 (10) 8 (2)** 5 (1)** Bacteroides < 1 < 1 < 1 < 1 0.4 (0.4)** 4 (3)** < 1 3 (2) 0.4 (0.2)** 2 (1)** Parabacteroides < 1 < 1 < 1 < 1 0.3 (0.3)** 4 (4)** < 1 2 (2) 0.3 (0.2)** 2 (2)** Prevotella – < 1 < 1 – 0.01 (0.01)** 0.3 (0.4)** < 1 < 1 –** 0.1 (0.1)** Odoribacter – – < 1 – < 1 < 1 < 1 < 1 0.4 (0.3)** 0.6 (0.5)** Proteobacteria 0.1 (0.1)* 1 (1)* < 1 1 (1) < 1 1 (2) 3 (3) 10 (16) 0.7 (0.4)*** 2 (2)*** Alphaproteobacteria – < 1 < 1 < 1 – < 1 < 1 1 (2) < 1 < 1 Betaproteobacteria < 1 < 1 < 1 < 1 < 1 < 1 1 (2) 5 (8) 0.2 (0.001)** 0.1 (0.04)** Deltaproteobacteria 0.003 (0.01)* 1 (1)* 0.021 (0.028)* 0.5 (0.5)* < 1 < 1 < 1 < 1 < 1 < 1 Desulfovibrio –** 1 (1)** 0.003 (0.006)** 0.5 (0.5)** –** 0.2 (0.2)** 0.002 (0.005)*** 0.3 (0.2)*** 0.002 (0.005)** 0.6 (0.3)** Epsilonproteobacteria – < 1 – < 1 –* 0.02 (0.1)* < 1 2 (5) –** 2 (2)** Unknown genus (family Helicobacteraceae) – < 1 < 1 < 1 –*** 1 (1)*** 0.002 (0.006) 2 (5) –*** 2 (2)*** Gammaproteobacteria < 1 < 1 < 1 < 1 < 1 < 1 < 1 1 (1) < 1 < 1 Actinobacteria < 1 < 1 < 1 < 1 < 1 < 1 < 1 < 1 0.4 (0.2)** 0.2 (0.1)** TM7 0.002 (0.006)* 0.2 (0.2)* –** 0.12 (0.14)** 0.003 (0.01)* 0.2 (0.1)* < 1 < 1 0.001 (0.003)* 0.2 (0.1)* Deferribacteres < 1 < 1 < 1 < 1 < 1 < 1 11 (11) 9 (10) < 1 < 1 Deferribacteres < 1 < 1 < 1 < 1 < 1 < 1 11 (11) 9 (10) < 1 < 1 Mucispirillum < 1 < 1 < 1 < 1 < 1 < 1 11 (11) 9 (10) < 1 < 1 Statistical tests of over-or under-representation of bacterial lineages among sample groups (Room 1 versus Room 2) at each sample location were made at the phylum (bold), class (bold) and genus levels using Wilcoxon's rank-sum test. To correct for multiple testing, the P-values were converted to false discovery rate values (Q-values). (–) corresponds to no sequences present. For values < 1, the exact value is only given if significantly different. *P-value < 0.001, Q-value < 0.001, **P-value <0.001, Q-value < 0.01, ***P-value < 0.01, Q-value< 0.05. Comparing mice in the two rooms at the class and genus levels revealed pronounced differences (Food A, Figs 1D–E and 2A–F, Table 1). In Room 1, the genus Anaerostipes, within the Clostridia class, was found in significantly higher amounts at all locations throughout the gut (Fig 2A, Table 1). The Erysipelotrichi class was also found in higher abundances at all locations in Room 1 (Fig 1E, Table 1). 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 (Fig 2B, Table 1). Within the Bacteroidia class, an unknown genus (family S24-7) was found at significantly higher levels in Room 1 in both ileum lumen and caecum (Table 1). Figure 2. Significant genera between the two mouse colonies A–F. The relative abundance (%) of the genera Anaerostipes (A), Allobaculum (B), Desulfovibrio (C), Bacteroides (D), Parabacteroides (E) and Prevotella (F) in ileum lumen Room 1 (n = 11) and Room 2 (n = 11), ileum mucus Room 1 (n = 11) and Room 2 (n = 11), distal colon lumen Room 1 (n = 11) and Room 2 (n = 11), distal colon mucus Room 1 (n = 9) and Room 2 (n = 7), and caecum Room 1 (n = 11) and Room 2 (n = 11). Data are presented as mean ± SD. Wilcoxon's rank-sum test was conducted to compare over- or under-representation of the different genera. To correct for multiple testing, the P-values were converted to false discovery rate values (Q-values). *P < 0.001, Q < 0.001, **P < 0.001, Q < 0.01, ***P < 0.01, Q < 0.05. Source data are available online for this figure. Source Data for Figure 2 [embr201439263-SourceData-Fig2.xlsx] Download figure Download PowerPoint In Room 2, the phylum TM7 was a small component but had a higher relative abundance at all locations (Fig 1C, Table 1). Proteobacteria was also generally more abundant in Room 2 (Supplementary Fig S4, Table 1). 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 (Fig 1D, Table 1). Within this class, an unknown genus of the Helicobacteraceae family was found at higher levels in Room 2 (Table 1). Within the Deltaproteobacteria, one genus, Desulfovibrio, was found in significantly higher amounts at all locations of mice in Room 2 (Fig 2C, Table 1). The Clostridia class was significantly more abundant in ileum mucus and caecum samples of Room 2 (Fig 1E, Table 1). 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 (Table 1). SFB also showed a small but significant difference between the rooms in the distal colon with higher levels in Room 2. Also, within the Bacteroidia class, the genera Bacteroides, Parabacteroides, and Prevotella were found in significantly higher abundances in distal colon lumen and caecum samples in Room 2 (Fig 2D–F, Table 1). The two diets used were tested by analysing animals in both locations also fed with Food B diet (autoclaved). Altering the diet from weaning gave some shift in the microbiota, but less than the difference between the two rooms. 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). The phylum TM7 was also found at higher levels at all gut locations and rooms when mice were fed Food B (Supplementary Table S1). The Erysipelotrichi class and the genus Allobaculum was found in higher relative abundances in ileum and distal colon, as well as in caecum when the mice were fed Food B in Room 1 (Supplementary Table S1). It was thus concluded that the two C57BL/6 mice strains bred separately had stable, but different microbiota that were vertically transmitted. Food influenced the microbiota to a small extent. This raised the question of the effect of the different microbiota on the mucus thickness and properties. Differences in the mucus quality in mice from the two husbandries The thickness of the secreted mucus from mice in the two rooms was measured on small intestinal and distal colon explants mounted in a perfusion chamber after visualization of the mucus surface by charcoal 21. 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 (Fig 3A). In colon, the mucus forms two layers; an outer non-attached and an inner mucus layer that is firmly attached to the epithelium 14. The thickness of the inner mucus layer as well as the mucus growth did not differ significantly between the two rooms on either food (Fig 3B). Figure 3. Differences in mucus properties in the two mouse colonies and in free-living mice Mucus thickness on explants from distal small intestine (pre) and mucus remaining after aspiration (post) in the different rooms (Room 1 and 2) with the different foods (Food A and Food B). Data are presented as mean ± SEM (n = 3). Mucus thickness measured during one hour on distal colon explants from mice in the two rooms (Room 1 and 2) with different foods (Food A and Food B). The mucus not attached to the epithelium was removed after 60 min and the attached mucus was measured (post). Data are presented as mean ± SEM (n = 3). Penetrability of beads (red 0.5 μm, purple 1 μm, green 2 μm) in mucus on explants from mice housed in Room 1 or Room 2 with either Food A or Food B. The epithelium is stained by Calcein violet (blue). Double arrows indicate impenetrable mucus. Scale bar is 100 μm. The impenetrable mucus as the mean distance of the 20 most penetrable beads shows a significant difference between the mice in the two rooms with Food A. Data are presented as boxplots with median and min–max whiskers. Mann–Whitney U-tests were performed to compare groups (Room 1 and 2) on each food (Food A: n = 5, Food B: n = 3–4, *P = 0.032). Mucus penetrability as in (C) on explants from free-living mice. Double arrow indicates impenetrable mucus. Scale bar is 100 μm. Immunostaining for Muc2 (green) of distal colon sections from free-living mice with Hoechst counterstain (blue). Double arrow indicates inner mucus layer without bacteria (visualized by DNA stain). Scale bar is 50 μm. Source data are available online for this figure. Source Data for Figure 3A, B, D [embr201439263-SourceData-Fig3A-B-D.xlsx] Download figure Download PowerPoint The size exclusion properties of the mucus can be assessed by allowing fluorescent beads, the size of bacteria, to sediment through the mucus formed on colonic explants 15. Using this system, the secreted mucus has previously been shown to separate the beads from the epithelium in both mouse and human biopsies. 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 (Fig 3C). Mice given food B showed increased penetrability in both groups, but a difference still remained between Room 1 and 2 mice (Fig 3C). The penetrability of beads was estimated as the distance from the epithelium to the 20 closest located beads (Fig 3D). This showed significantly more penetrable mucus with a thinner, impervious part of the inner mucus layer in Room 2. Mice in Room 2 thus differed in mucus properties compared to mice in Room 1 that we had assumed to have a normal mucus layer. This raised doubts on what is a normal mucus layer. To address this, mice caught in their wild habitat were studied with the same methods as for mice from the two rooms. In free-living mice, we could typically observe a non-penetrable inner colon mucus layer that separated the beads from the epithelium even further than what was observed in mice from Room 1 (Fig 3E). Tissue staining of distal colon tissue could also show bacteria well separated from the epithelium (Fig 3F). 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. Bacteria penetration in mucus and histology of mice in different husbandries Muc2 mucin immunohistochemistry revealed that the mucus in fixed sections looked different in mice from Room 2 on Food A (Fig 4A). 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. A good separation of bacteria and epithelium by a stratified inner mucus layer was only present in mice from Room 1 (Fig 4A). 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 (Fig 4B). Figure 4. Bacteria localization and histology of mice from the two husbandries A, B. Immunostaining of colon sections from mice in the two rooms with Food A (A) or Food B (B) using Anti-MUC2C3 (green) and FISH with a general bacterial 16S rRNA gene probe to detect bacteria (red) counterstained with Hoechst (blue). Double arrows mark the inner mucus layer that separates bacteria from the epithelium. Bacteria are found within the inner mucus layer (indicated by arrows) in mice housed in Room 2. Scale bar is 10 μm. C. Immunostaining of the Muc2 precursor before it gets O-glycosylated using the anti-apoMuc2 antiserum on mice from both rooms fed Food A. Scale bar is 100 μm. D. The crypt length was measured in distal colon on sections from mice in the two rooms on Food A (n = 4) E. The number of goblet cells per crypt was counted in distal colon of mice in the two rooms on Food A (n = 10). F. The numb","datePublished":"2024-10-05T18:49:52.589844Z","author":[{"@type":"Person","name":"Hedvig Jakobsson","url":"/author/1558115"},{"@type":"Person","name":"Ana Rodríguez‐Piñeiro","url":"/author/1616695"},{"@type":"Person","name":"A.H. Schutte","url":"/author/1676731"},{"@type":"Person","name":"Anna Ermund","url":"/author/1136228"},{"@type":"Person","name":"Preben Boysen","url":"/author/4406165"},{"@type":"Person","name":"Mats Bemark","url":"/author/1245488"},{"@type":"Person","name":"Felix Sommer","url":"/author/1147756"},{"@type":"Person","name":"Fredrik Bäckhed","url":"/author/1467388"},{"@type":"Person","name":"Gunnar Hansson","url":"/author/1095130"},{"@type":"Person","name":"Malin Johansson","url":"/author/3972979"}],"publisher":{"@type":"Organization","name":"ResearchHub","logo":{"@type":"ImageObject","url":"https://researchhub.com/logo.png","width":512,"height":512},"url":"https://researchhub.com"},"inLanguage":"en-US"}

Paper

Paper