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Articles in PresS. Am J Physiol Gastrointest Liver Physiol (July 5, 2013). doi:10.1152/ajpgi.00046.2013 1 Gastrointestinal mucus layers have different properties depending on location - 1. Studies of mucus in mouse stomach, small intestine, Peyer´s patches and colon Anna Ermund, André Schütte, Malin E.V. Johansson, Jenny K. Gustafsson, and Gunnar C. Hansson* Department of Medical Biochemistry, University of Gothenburg, Gothenburg, Sweden. RUNNING HEAD: Mouse Gastrointestinal Mucus Properties Address for reprint requests and other correspondence: Gunnar C. Hansson, Dept. Medical Biochemistry, University of Gothenburg, Box 440, 40530 Gothenburg, Sweden; e-mail: [email protected]; Phone: +46 31 7863488; Fax: +46 31 416108. Copyright © 2013 by the American Physiological Society. 2 Abstract Colon has been shown to have a two-layered mucus system where the inner layer is devoid of bacteria. However, a complete overview of the mouse gastrointestinal mucus system is lacking. We now characterize mucus release, thickness, growth over time, adhesive properties and penetrability to fluorescent beads from stomach to distal colon. Colon displayed spontaneous mucus release and all regions released mucus in response to carbachol and PGE2, except the distal colon and domes of Peyer´s patches. Stomach and colon had an inner mucus layer that was adherent to the epithelium. In contrast, the small intestine and Peyer´s patches had a single mucus layer that was easily aspirated. The inner mucus layer of the distal colon was not penetrable to beads the size of bacteria and the inner layer of the proximal colon was only partly penetrable. In contrast, the inner mucus layer of stomach was fully penetrable, as was the small intestinal mucus. This suggest a functional organization of the intestinal mucus system where the small intestine has loose and penetrable mucus that may allow easy penetration of nutrients in contrast to the stomach where the mucus provides physical protection and the colon where the mucus separates bacteria from the epithelium. This knowledge of the mucus system and its organization improves our understanding of the gastrointestinal tract physiology. 227 words Key words: Mucin, goblet cells, bacteria, mucus adhesiveness, mucus thickness. 3 INTRODUCTION The gastrointestinal tract (GIT) is subject to massive and continuous physical as well as chemical assault from ingested food and in order to extract nutrients from it, hydrochloric acid, digestive enzymes and bile acids are released, together creating a potentially harsh environment for the single layer of columnar epithelial cells (4; 28). Although this varies by region, food and luminal contents contain large numbers of diverse microorganisms which can be potentially harmful if they breach the mucus or epithelial barrier (12). For protection, the gastrointestinal epithelium is covered by mucus where the main constituent is the secreted gel forming mucins; in the stomach MUC5AC and in the intestine MUC2, that are also the two most similar of the secreted mucins (15). Mucins are large glycoproteins where the glycans make up more than 80% of the molecular mass. The central parts have a protein backbone containing sequences rich in proline, threonine and serine, the so-called PTS-sequences, which are highly O-glycosylated and constitute the mucin domains. The gel-forming mucin MUC2 oligomerizes into large net-like polymers when the C- and N-termini form disulfide-bond stabilized di- and trimers (1). Mucins are produced, stored and released by specialized cells called goblet cells, characterized by their distended theca containing mucin granules (15). The number of goblet cells relative to enterocytes increases from the proximal to distal intestine, constituting 4%, 6%, 12%, and 16% in the duodenum, jejunum, ileum, and distal colon, respectively (19). There are relatively few bacteria residing in the stomach and the proximal small intestine (5), but the number of bacteria increases distally where the distal ileum has approximately 108 bacteria per ml of luminal content and colon 1011. This large amount of bacteria is kept separated from the epithelium by an inner mucus layer physically impenetrable to bacteria (16). The bacteria on the other hand reside and thrive in the outer mucus layer (15). The host benefits from harboring bacteria as they degrade the mucins and 4 ingested complex carbohydrates from the food into simple sugars that are converted into butyrate, propionate and acetate supplied to the host (3). Antigens in the bowel content are sampled by lymphoid follicles, which are part of the gut-associated lymphoid tissues of the adaptive immune system (12). In the small intestine the aggregated lymphoid follicles are organized in Peyer´s patches, consisting of domes with large numbers of immune cells underneath a single epithelial cell layer called the follicle-associated epithelium (FAE). Interspersed among the FAE enterocytes are the M cells (6; 22), cells specialized for endocytosis and capable of passing antigens from the gut lumen to the underlying immune cells. Recently it has also been suggested that goblet cells can act as sampling sites for dendritic cells (20). Although much is known about the immune cells in the Peyer´s patches, it is debated whether the domes of Peyer´s patches are covered by mucus (21; 24). The gastrointestinal mucus system has been relatively poorly explored and understood as it is essentially invisible and collapses upon common formaldehyde fixation. A large step forward was taken by Atuma et al, who could visualize the luminal surface of the mucus by adding charcoal particles and measure the mucus thickness (2). These studies were made in rats, but mice are commonly used for studies of gastrointestinal physiology and pathophysiology as these can be easily genetically manipulated. We have developed a methodology and performed studies on the mucus system of the distal colon (10; 16; 17), but to provide a more complete picture of gastrointestinal mucus system we are now characterizing the normal mucus system of the whole GIT. Here we characterize the mucus system from stomach to distal colon, including Peyer´s patches as mucus release mucus thickness, growth over time, adhesive properties and penetrability to fluorescent beads in C57BL/6 mice. 5 MATERIAL AND METHODS Animals. All animal procedures were approved by the local Laboratory Animal Ethics Committee, Gothenburg, Sweden. Mice were kept under specific pathogen free conditions in individually ventilated cages under controlled temperature (21–22°C), humidity and 12 h light/dark cycle. They were given standard chow and water ad libitum. Male and female C57BL/6 mice (age 8-16 weeks) were euthanized by isoflurane and cervical dislocation. Explant Tissue. Gastrointestinal tissue was dissected, flushed with ice cold oxygenated (95% O2, 5% CO2) Krebs transport buffer and mounted in horizontal open Ussing-type chambers a with 4.9 mm2 circular opening as described previously (10). To avoid disturbing the mucus gel, the apical chamber was kept unstirred with a constant volume. Mucus release was stimulated by perfusing the explants basolaterally with a combination of the secretagogues carbachol and prostaglandin E2 (PGE2), 10 µM of each for 40 min. Mucus Thickness Measurements. Mucus measurements and video recordings were performed as described previously (9; 10). Mucus was aspirated with a Gilson Pipetman® P200 (Middleton, WI) set to 150 µl and a yellow tip (no. 70.760.502, Sarstedt, Nümbrecht, Germany). In the small intestine, mucus was measured from the mucus surface to the villi tips, and after mucus was removed and villus height was measured from the epithelium between the villi to the villi tips. Total mucus thickness is presented as the sum of these two measurements. To evaluate thickness of easily aspirated mucus, the whole apical volume was aspirated using a plastic Pasteur pipette (PP-101, outer tip diameter 0.9 mm, inner tip diameter 0.7 mm, max volume 800 µl, Cellprojects, Sutton Valence, UK) during approximately three seconds. The remaining mucus thickness (denoted “Post”) was measured 6 after refilling the apical chamber with 150 µl Krebs-mannitol and the addition of new charcoal particles. Mucus Penetrability. Explants from stomach, small intestine or colon were mounted in a horizontal imaging chamber and recorded as described previously (10; 13). The chamber was heated to 37°C during a period of 10 min and the apical buffer was removed and a suspension of 0.5 µm (red), 1 µm (far red) and 2 µm (green) fluorescent beads (FluoSpheres, Invitrogen, Carlsbad, CA) was added. The beads were allowed to settle in the mucus for 5 min before new Krebs-mannitol buffer was added and the beads left to sediment for 40 min. Confocal Z-stacks (optical section 2.8 µm, interval 10 µm) were taken to analyze the distribution of the different beads throughout the mucus, using an upright LSM 700 Axio Examiner 2.1 confocal imaging system with a Plan-Apochromat ×20/1.0DIC water objective (Carl Zeiss, Oberkochen, Germany). Volocity 6.0 (PerkinElmer, Waltham, MA) software was used to process images. Histology and immunostaining. Stomach tissue was fixed in Karnovsky fixative (2% paraformaldehyde, 2.5% glutaraldehyde in 0.05 M, sodium cacodylate buffer, pH 7.2) for 24 h followed by sequential staining using 1% OsO4 for 4 h, 1% tannic acid for 3h and 1% Uranyl acetate overnight. Samples were dehydrated and embedded in epoxy resin (Agar 100, Agar Scientific, Stansted, UK). Sections (1 µm) were stained with Periodic acid-Schiff. Whole tissue from stomach, duodenum, jejunum, ileum, proximal and distal colon with content was fixed in methanol-Carnoy’s fixative and stained after antigen retrieval (16) with the anti-MUC2C3 antiserum raised against a peptide from a non-glycosylated region (1:500) (16) or mouse monoclonal anti-MUC5AC (45M1, 1:2000; Invitrogen, Carlsbad, CA). As secondary antibodies goat anti-rabbit Alexa 488 or goat anti-mouse Alexa 488 (Invitrogen, Carlsbad, CA) were used. DNA was stained by TO-PRO®-3 Iodide (1 µM, 642⁄661, Invitrogen, Carlsbad, CA) or DAPI (Invitrogen, Carlsbad, CA). Images were acquired using a 7 fluorescence microscope, Eclipse E1000 with a Plan-Fluor 40x/0.75 DIC objective (Nikon, Amstelveen, The Netherlands) or an upright LSM 700 Axio Examiner.Z1 laser scanning confocal microscope, with a Plan-Apochomat 40x/1.3 Oil DIC objective (Zeiss, Oberkochen, Germany) The pictures were processed uniformly using the ZEN 2010 software (Zeiss, Oberkochen, Germany), Volocity 6.0 and Adobe Photoshop. Drugs. Carbachol and prostaglandin E2 (PGE2, Sigma, St Louis, MO), were dissolved in water or a 1:1 mixture of ethanol and DMSO at 10-1 M, respectively. Substances were then further diluted in Krebs-glucose buffer. Statistical Analysis. Data are presented as mean ± standard error of the mean (SEM) for n animals. Mann-Whitney test was used to test differences between two groups. Statistical significance was accepted when P < 0.05. RESULTS AND DISCUSSION Colonic Mucus To study the mucus in mouse proximal and distal colon, explants were mounted in a horizontal perfusion chamber and viewed through a stereomicroscope (10). The transparent mucus was visualized by charcoal particles sedimented onto the mucus surface (Fig. 1A). The macroscopic morphology of the proximal colon is different from the distal as the proximal part has diagonal striations that are observed as epithelial folds in the microscope. After mounting, the outer mucus layer was aspirated and the remaining mucus thickness measured (time 0, Fig. 1B, C). This thickness represents the inner colon mucus layer (16). The mucus was then allowed to grow and the thickness measured after 20, 40, and 60 min. During this hour, the mucus in proximal colon increased by 75±19 µm and in the distal colon by 87± 14 µm. After this, the outer mucus was aspirated again (after removal, Fig. 1B) and the inner mucus layer measured 37±2.4 µm for the proximal and 44±3.9 µm for the distal colon. The 8 inner mucus layer thicknesses of these two sections recorded at time 0 and after removal at 60 was identical, indicating that the outer layer is formed from the inner mucus and that the thickness of this inner layer is maintained constant. To illustrate that the outer mucus layer can be aspirated in both the proximal and distal colon, movies showing the mucus aspiration were recorded (supplemental videos 1 and 2, initial frames are shown in Fig. 1A). A combination of carbachol and PGE2 has been shown to stimulate mucus release, especially in the small intestine (10; 11; 25; 30). To test if it is possible to stimulate colon mucus secretion, explants were stimulated with carbachol and PGE2 and mucus thickness measured every 20 min (Fig. 1C). The mucus thickness increase in the stimulated explants was 185±21 µm in the proximal colon (P = 0.04, *) and 113±18 µm in the distal colon (P = 0.59, n.s.). Thus stimulation caused increased mucus thickness (2.5 times) in the proximal, but not in the distal colon. Fixed tissue sections from proximal and distal colon were stained for Muc2 using the anti-MUC2C3 antibody (green) and nuclei (DAPI, blue, Fig. 1D). The inner layer (i) can be observed in both the proximal (left) and distal (right) colon, but generally appears thicker and more continuous in the distal colon. The inner layer in both proximal and distal colon looks stratified. The inner mucus layer in distal colon does not show any DNA staining indicative of bacteria. This layer thus acts as a barrier limiting bacterial exposure to the epithelium. In the proximal colon, the innermost mucus separates the epithelium from most bacteria. Previously we have shown that beads the size of bacteria do not penetrate the distal inner mucus layer (10; 13). To study the penetrability of beads also in the proximal colon, explants were mounted in the horizontal Ussing-type chamber and fluorescent beads with diameters of 0.5 (red), 1 (purple) and 2 (green) µm were allowed to sediment into the mucus for 40 min, after which confocal Z-stacks were acquired (Fig. 1E, tissue stained blue). 9 Three different conditions are shown: (1) between folds, (2) on top of a fold and (3) after stimulation with carbachol and PGE2 (10 µM each) for 10 min. The mucus penetrability in the proximal colon thus varied depending on where the Z-stacks were acquired. Between the folds, the mucus accumulated and was impenetrable to beads, whereas on top of the folds the mucus tended to form a thinner and more penetrable layer. Penetrability of secretagogue stimulated, newly secreted mucus was not different from spontaneously released mucus in the proximal colon (Fig. 1E). In the distal colon, the distance separating the lowest beads and the stained tissue (blue) was around 200 μm (Fig. 1F). Thus the inner layer measured as bead separation was considerably thicker than was measured with the charcoal method. Although it is not fully understood how the inner mucus layer of the colon becomes impenetrable to bacteria (16), our studies using fluorescent beads with sizes typical for bacteria suggest that the inner mucus layer is acting as a molecular sieve (10; 13). In fact, our current experience is that bead penetrability in the colon directly reflects bacterial penetrability. There is, however, a discrepancy in the thickness of the inner mucus layer measured by the charcoal method compared to the thickness of the mucus impenetrable to fluorescent beads measured in the confocal Z-stacks. These should both correspond to mucus impenetrable to bacteria. The reason for this difference is unknown, but we speculate that the outer layer, although it is released from its attachment to the cells, is still denser close to the inner-outer transition. Small intestinal mucus. Duodenal, jejunal and ileal explants were mounted in the horizontal chamber and charcoal particles added to visualize the transparent mucus (Fig. 2A). The mucus was then aspirated as shown in the movies (supplemental video 3, duodenum; video 4, jejunum; video 5, ileum, initial frames are shown in Fig. 2A). The charcoal was initially localized on the mucus surface 10 above the villi, which can be observed protruding toward the viewer through the mucus. The duodenum and jejunum mucus was intermixed with fat droplets and other food particles. The foodstuff could not be removed before mucus thickness measurements and thus the measured mucus thickness in the proximal parts of the small intestine, represents both mucus and intermixed food. The mucus was more homogeneous in the more distal parts. The mucus in all three parts can be easily aspirated with a pipette and charcoal added after mucus removal sediments down between the villi and comes close to the crypt openings, indicating that there was no mucus left. In contrast to the colon, charcoal particles could sediment through the mucus (after 20 min). The small intestinal mucus network built by the Muc2 mucin polymers is more sparse than in the colon showing that the same Muc2 gene product can build mucus with remarkably different properties. Small intestinal explants do not spontaneously secrete mucus when mounted in the chamber (Fig. 2B), in contrast to colonic explants. However, upon stimulation with carbachol and PGE2 (Fig. 2C, arrow), new mucus is secreted and after 40 min it almost reaches the same thickness as before removal, mucus being significantly thicker after stimulation compared to unstimulated samples (Fig. 2C).This mucus can be aspirated in all sections (data not shown). Immunostainings for Muc2 (green) in Carnoy fixed tissue sections from duodenum, jejunum and ileum, revealed thin, thread-like mucus between the villi (Fig. 2D, duodenum (1), jejunum (2) and ileum (3)). The Muc2-stained mucus does not fill the space between the villi, likely an artifact due to shrinking of the mucus gel during fixation and processing. In most instances, the mucus covered the villi tips. DNA-stained bacteria (blue; arrows in Fig. 2, duodenum (E), jejunum (F) and ileum (G)) could be observed in the Muc2stained mucus (green) especially in the distal small intestine. The bacteria were found in 11 proximity to the villi tips, but seldom in direct contact with the epithelium. Very few bacteria were found between the villi, and no bacteria were observed close to the crypt openings. The penetrability to fluorescent beads was tested as for the colonic explants. The ileal mucus was completely penetrable to the fluorescent beads as shown in Fig. 2H. The tall villi in the duodenum and jejunum do not stand perpendicular to the tissue when mounted in the chamber and consequently the beads do not easily sediment down between the villi. Thus it was not possible to obtain a correct picture of mucus penetrability in these sections, but whenever the beads were not stopped by tilted villi they penetrated the mucus. We thus conclude that the mucus in all parts of the small intestine was penetrable to beads the size of bacteria. A majority of the nutrients from ingested food are taken up in the small intestine and it is thus not difficult to accept that the small intestinal mucus is different from that in colon. Instead of a physical barrier as in colon, the bacteria are likely kept at bay by the antibacterial peptides and proteins secreted by the epithelial cells and the Paneth cells in the crypts (4; 31). It can be suggested that the mucus decreases the diffusion rate of both bacteria and antibacterial compounds and consequently generates a gradient limiting the contact between bacteria and the epithelium (14). The small intestinal mucus fills out most of the space between the villi and usually also covers the villi tips. However, bacteria can be observed relatively close to the villi tips, especially in the distal small intestine. In the ileum, the mucus is denser and forms a more continuous mucus layer than in more proximal parts. We observed very few bacteria between the villi and never close to the crypt openings. Most likely this is because most of the mucins are secreted at the crypt opening and as this is mixed with the antibacterial peptides and proteins (e.g. defensins, lysozyme, and DMBT1 (deleted in malignant brain tumors 1)) from the Paneth cells, there is a flow of mucus that efficiently hinders bacteria from entering the crypts. 12 The fact that the small intestinal mucus is easy to aspirate and remove is in contrast to mice with a non-functional Cftr ion channel (10) where the mucus is attached. This was attributed to the bicarbonate transporting property of the Cftr channel as the attached mucus was detached when secreted into buffers with high amounts of bicarbonate. Mice with a non-functional Cftr channel show distal ileal obstruction and ileal bacterial overgrowth (7; 8; 23). This is similar to cystic fibrosis patients who frequently have intestinal obstruction problems (DIOS, distal intestinal obstruction syndrome). This illustrates the importance of having non-attached mucus that can trap bacteria and move these distally with the fast small intestinal peristalsis. Mucus on Peyer´s patches. To address if Peyer´s patches are covered with mucus, mouse ileal explants containing Peyer´s patches were mounted in the horizontal chamber and movies illustrating the gentle aspiration of mucus visualized by charcoal were recorded. The epithelial surfaces of all analyzed Peyer’s patches were covered with mucus as shown in Fig. 3A and corresponding video (Supplemental video 6, initial frame in Fig. 3A). Four domes are found in this video with villi in-between. The presence of mucus is evident from the charcoal localized above the dome epithelium and the fact that aspiration can remove the charcoal containing mucus. Charcoal added after aspiration sedimented down onto the epithelium, suggesting that all mucus was removable. Like mucus in the small intestine, the mucus on Peyer´s patches was penetrable to fluorescent beads (Fig. 3B, 40 min sedimentation). Most beads sedimented to the epithelium, but some remain suspended in the mucus, further supporting the notion that there is a mucus layer on top of the domes. It has been controversial whether there is mucus on top of Peyer´s patches. When we analyzed our explants for mucus we observed, as has been done previously in rats 13 (18), that there is mucus on top of the patches. As this mucus is readily removable and also penetrable to beads the size of bacteria, this mucus will not hinder bacteria from reaching the domes. Stomach mucus. Mouse stomach corpus was mounted in the perfusion chamber (Fig. 4A, right) and the mucus surface visualized with charcoal. As shown in the supplemental video 7, only small amounts of the charcoal could be removed by gentle aspiration and also most of the outer mucus is still attached. This is in contrast to the colon where the outer layer is easily removed. The inner mucus layer was firmly adherent, as illustrated in supplemental video 8 (Fig. 4A, left). The inner mucus layer can only be removed after applying strong force that damaged the epithelium. This suggests that the inner mucus layer of the stomach is more firmly attached to the epithelium than in the colon. Mucus thickness was measured after mounting the stomach explants in the chamber and after the outer mucus was aspirated. Remaining mucus thickness, corresponding to the inner mucus layer, was 45±3 µm directly after removal (time 0, Fig. 4B), a thickness almost identical to in vivo measurements (27). No spontaneous increase in mucus thickness was observed during the 60 min (open circles), but when the tissue was stimulated with carbachol and PGE2 (arrow), the mucus thickness increased to 88±11 µm (Fig. 4B; closed circles), mucus being significantly thicker after stimulation than in unstimulated explants. The stomach mucus thickness was measured after stimulation of mucus release (Fig. 4C, Pre) and then after gentle aspiration (Fig. 4C, Post). No mucus could be removed, illustrating that stomach mucus is firmly attached to the epithelium also after secretion and not converted into easily aspirated mucus as in the colon. Light microscopy of Karnovsky fixed, epoxy resin embedded and PAS stained mouse stomach showed a thin, poorly stained, striated layer on top of the epithelium (Fig 4D, 14 arrow). This striated structure resembles the colonic inner mucus layer, but is very likely considerably dehydrated and maybe also less organized. Carnoy fixed paraffin sections of the stomach corpus stained for the Muc5ac mucin (green) and DNA (blue) show Muc5ac positive cells along the surface and at the upper parts of the glands. Attached mucus was also observed on the epithelial surface (Fig. 4E). The stomach mucus was also tested for bead penetrability. As the normal pH of the stomach is low, three different experiments were performed: 1) Beads were directly applied on the mucus present on the tissue at pH 7.4 (Fig. 4F-1); 2) Mucus secretion was stimulated with carbachol and PGE2, beads applied at pH 7.4 (Fig. 4F-2); 3) Secretion was stimulated into an apical pH 3 buffer to mimic stomach conditions (Fig. 4F-3). Irrespective of experimental conditions, beads penetrated the mucus and reached the epithelial cell surface. Therefore, in contrast to the colon mucus, the stomach mucus is penetrable to fluorescent beads the size of bacteria. In the stomach, the mechanical stress is high as the ingested food is coarse, with large, hard, undigested pieces that can cause mechanical damage together with the chemical challenge caused by the gastric acid, but the bacterial load is comparatively low. The stomach has, similar to the colon, mucus that is firmly attached to the epithelium. This point is clearly demonstrated in supplemental videos 7 and 8. The inner mucus act as a slow diffusion barrier as it helps create a pH gradient from 1-2 in the lumen to 7 close to the epithelium, by limiting the diffusion of luminal hydrochloric acid and epithelial bicarbonate (26; 29). Our experiments also show that the stomach is relatively stable at low pH, as the mucus formed in apical buffer with pH 7.4 or 3 had similar attachment properties and penetrability. Conclusions 15 The separation of bacteria from the epithelium by a non-penetrable inner mucus layer was previously described for the distal colon (16). Here we demonstrated that the proximal colon also has an inner mucus layer, but this layer is partly penetrable to beads the size of bacteria. The stomach like colon has an attached mucus layer, whereas the small intestine only have an easily removable mucus. Thus the mucus of the alimentary tract has fundamentally different secretory mechanisms, appearance and properties. The two-layered mucus system of colon is also present in germ-free mice (16), but less developed with thinner thickness. Small intestinal mucus differ in being thinner, non-adherent, and more penetrabel indicating differences in the barrier to bacteria. ACKNOWLEDGEMENT We acknowledge the Electron Microscopy Unit and the Centre for Cellular Imaging at the University of Gothenburg for technical help. GRANTS This work was supported by the Swedish Research Council, The Swedish Cancer Foundation, The Knut and Alice Wallenberg Foundation, IngaBritt and Arne Lundberg Foundation, Sahlgren's University Hospital (LUA-ALF), Wilhelm and Martina Lundgren’s Foundation, Torsten och Ragnar Söderbergs Stiftelser, The Sahlgrenska Academy, National Institute of Allergy and Infectious Diseases (U01AI095473, the content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH), and The Swedish Foundation for Strategic Research - The Mucus-Bacteria-Colitis Center (MBC) of the Innate Immunity Program. DISCLOSURES 16 The authors have nothing to disclose. AUTHOR CONTRIBUTIONS AE, AS, JKG and MEVJ performed experiments. AE, AS, JKG and MEVJ analyzed data. AE, JKG, MEVJ and GCH designed experiments. AE and GCH wrote this manuscript. REFERENCES 1. Ambort D, Johansson MEV, Gustafsson JK, Nilsson H, Ermund A, Johansson BR, Kock P, Hebert H and Hansson GC. Calcium and pH-dependent Packing and Release of the Gel-forming MUC2 Mucin. Proc Natl Acad Sci U S A 109: 5645-5650, 2012. 2. Atuma C, Strugula V, Allen A and Holm L. The adherent gastrointestinal mucus gel layer: thickness and physical state in vivo. Am J Physiol 280: G922-G929, 2001. 3. Backhed F, Ley RE, Sonnenburg JL, Peterson DA and Gordon JI. Host-Bacterial Mutualism in the Human Intestine. Science 307: 1915-1920, 2005. 4. Bevins CL. Paneth cell defensins: key effector molecules of innate immunity. Biochem Soc Trans 34: 263-266, 2006. 5. Bik EM, Eckburg PB, Gill SR, Nelson KE, Purdom EA, Francois F, Perez-Perez G, Blaser MJ and Relman DA. Molecular analysis of the bacterial microbiota in the human stomach. Proc Natl Acad Sci U S A 103: 732-737, 2006. 6. Corr SC, Gahan CC and Hill C. M-cells: origin, morphology and role in mucosal immunity and microbial pathogenesis. FEMS Immunol Med Microbiol 52: 2-12, 2008. 7. Delisle RC, Roach EA and Norkina O. Eradicaion of small intestinal bacterial overgrowth in the cystic fibrosis mouse reduces mucus accumulation. J Ped Gastroent Nutrition 42: 46-52, 2006. 17 8. French PJ, vanDoorninck JH, Peters RHPC, Verbeek E, Ameen NA, Marino CR, deJonge HR, Bijman J and Scholte BJ. A Delta F508 mutation in mouse cystic fibrosis transmembrane conductance regulator results in a temperature-sensitive processing defect in vivo. J Clin Invest 98: 1304-1312, 1996. 9. Gustafsson JK, Ermund A, Ambort D, Johansson MEV, Nilsson HE, Thorell K, Hebert H, Sjovall H and Hansson GC. Bicarbonate and functional CFTR channel is required for proper mucin secretion and link Cystic Fibrosis with its mucus phenotype. J Exp Med 209: 1263-1272, 2012. 10. Gustafsson JK, Ermund A, Johansson MEV, Schutte A, Hansson GC and Sjovall H. An ex vivo method for studying mucus formation, properties and thickness in human colonic biopsies and mouse small and large intestinal explants. Am J Physiol Gastrointest Liver Physiol 302: G430-G438, 2012. 11. Gustafsson JK, Sjovall H and Hansson GC. Ex vivo measurements of mucus secretion by colon explants. Methods Mol Biol 842: 237-243, 2012. 12. Hooper LV, Littman DR and Macpherson AJ. Interactions Between the Microbiota and the Immune System. Science 336: 1268-1273, 2012. 13. Johansson MEV, Gustafsson JK, Sjoberg KE, Pettersson J, Holm L, Sjovall H and Hansson GC. Bacteria penetrate the inner mucus layer before inflammation in the Dextran sulfate colitis model. PLoS ONE 5: e12238, 2010. 14. Johansson MEV and Hansson GC. Keeping Bacteria at a Distance. Science 334: 182183, 2011. 15. Johansson MEV, Holmen Larsson JM and Hansson GC. The two mucus layers of colon are organized by the MUC2 mucin, whereas the outer layer is a legislator of hostmicrobial interactions. Proc Natl Acad Sci USA 108: 4659-4665, 2011. 18 16. Johansson MEV, Phillipson M, Petersson J, Holm L, Velcich A and Hansson GC. 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. 17. Johansson MEV, Thomsson KA and Hansson GC. Proteomic Analyses of the Two Mucus Layers of the Colon Barrier Reveal That Their Main Component, the Muc2 Mucin, Is Strongly Bound to the Fcgbp Protein. J Proteome Res 8: 3549-3557, 2009. 18. Khan J, Iiboshi Y, Cui L, Wasa M and Okada A. Role of intestinal mucus on the uptake of latex beads by Peyer's patches and on their transport to mesenteric lymph nodes in rats. JPEN J Parenter Enteral Nutr 23: 19-23, 1999. 19. Kim YS and HO SB. Intestinal Goblet Cells and Mucins in Health and Disease: Recent Insights and Progress. Curr Gastroenterol Rep 12: 319-330, 2010. 20. McDole JR, Wheeler LW, McDonald KG, Wang B, Konjufca V, Knoop KA, Newberry RD and Miller MJ. Goblet cells deliver luminal antigen to CD103+ dendritic cells in the small intestine. Nature 483: 345-349, 2012. 21. McGuckin MA, Lind+¬n SK, Sutton P and Florin TH. Mucin dynamics and enteric pathogens. Nat Rev Micro 9: 265-278, 2011. 22. Neutra MR, Frey A and Kraehenbuhl JP. Epithelial M cells: Gateways for mucosal infection and immunization. Cell 86: 345-348, 1996. 23. Norkina O, Burnett TG and Delisle RC. Bacterial Overgrowth in the Cystic Fibrosis Transmembrane Conductance Regulator Null Mouse Small Intestine. Infect Immunity 72: 6040-6049, 2004. 24. Onori P, Franchitto A, Sferra R, Vetuschi A and Gaudio E. Peyer's patches epithelium in the rat: a morphological, immunohistochemical, and morphometrical study. Dig Dis Sci 46: 1095-1104, 2001. 19 25. Phillips TE, Phillips TH and Neutra MR. Regulation of intestinal goblet cell secretion. III. Isolated intestinal epithelium. Am J Physiol 247: G674-G681, 1984. 26. Phillipson M, Atuma C, Henriksnas J and Holm L. The importance of mucus layers and bicarbonate transport in preservation of gastric juxtamucosal pH. Am J Physiol 282: G211-G219, 2002. 27. Phillipson M, Johansson MEV, Henriksnäs J, Petersson J, Gendler SJ, Sandler S, Persson AEG, Hansson GC and Holm L. The gastric mucus layers: constituents and regulation of accumulation. Am J Physiol Gastrointest Liver Physiol 295: G806-G812, 2008. 28. Pott J and Hornef M. Innate immune signalling at the intestinal epithelium in homeostasis and disease. EMBO Rep 13: 684-698, 2012. 29. Schade C, Flemstrom G and Holm L. Hydrogen ion concentration in the mucus layer on top of acid-stimulated and -inhibited rat gastric mucosa. Gastroenterology 107: 180188, 1994. 30. Specian D and Neutra MR. Mechanism of rapid mucus secretion in goblet cells stimulated by acetylcholine. J Cell Biol 85: 626-640, 1980. 31. Vaishnava S, Yamamoto M, Severson KM, Ruhn KA, Yu X, Koren O, Ley R, Wakeland EK and Hooper LV. The Antibacterial Lectin RegIIIg Promotes the Spatial Segregation of Microbiota and Host in the Intestine. Science 334: 255-258, 2011. 20 Figure Legends Fig. 1. Colonic mucus forms two layers where the inner is a barrier to bacteria. (A) Explant tissue from the proximal (left) and distal (right) colon mounted in the perfusion chamber with the mucus surface visualized by charcoal and mucus aspirated (supplemental video 1 and 2, first frame in A). (B) Mucus thickness measuredin colon explants from proximal (white bars; n = 10) and distal (black bars; n = 10). Time 0; initial inner mucus layer. At 60 min, mucus was aspirated and the inner mucus layer thickness measured (after removal). (C) Mucus thickness measured in proximal (PC) and distal colon (DC), outer loose mucus removed (inner mucus layer, time 0), and mucus thickness measured (20 min). Paired explants, unstimulated (n=6) or stimulated (n=6) with carbachol and PGE2 (10 µM of each; arrow) and mucus thickness measured every 20 min. The difference between stimulated and unstimulated mucus thickness was significant in the proximal colon (P = 0.04), but not the distal colon (P = 0.59). (D) Immunostaining of Carnoy fixed proximal (left) and distal (right) colon for Muc2 (anti-MUC2C3 and Alexa 488 anti-rabbit Ig, green) and DNA (DAPI, blue). Inner mucus layer (i) excludes bacteria (blue, DAPI), but in the outer mucus layer (o). (E) Representative confocal Z-stacks of explants (tissue, blue) of proximal colon (n = 7) showing penetrability of mucus to fluorescent beads (red, 0.5 µm; purple, 1 µm; green, 2 µm). (E1) Between folds. (E2) On top of folds. (E3) Explant stimulated with carbachol and PGE2 (10 µM of each) to induce release of new mucus. Uneven mucus with penetrable and impenetrable region (arrow). (F) Representative confocal Z-stack visualizing penetrability to fluorescent beads, distal colon. Bar in A = 0.5 mm and bars in D, E and F = 50 µm. Fig. 2. Small intestine has one type of mucus, which is not attached. (A) Explant tissue from the duodenum (left), jejunum (middle) and ileum (right) mounted and the transparent mucus 21 visualized with charcoal and mucus aspirated showing non-attached mucus (supplemental video 3, 4 and 5, respectively). Mucus induodenum and jejunum was intermixed with food particles and lipid droplets. (B, C) Mucus thickness of paired explants from the duodenum, jejunum and ileum measured (n = 6) every 20 min (B) or stimulated with carbachol and PGE2 (10 µM of each; arrow in C). Stimulation gave a significantly thicker mucus layer in all segments, P = 0.0022, 0.0022 and 0.004 (**) in duodenum, jejunum and ileum, respectively. After the initial mucus thickness measurement, mucus was removed and remaining mucus thickness measured (both time 0). (D) Immunostainings of Carnoy fixed tissue sections for Muc2 (anti-MUC2C3 and Alexa 488 anti-rabbit Ig, green) and DNA (DAPI, blue). Due to mucus shrinkage caused by fixation and staining, the mucus appears thread-like. (E, F, G) Bacterial DNA (blue; arrows) and Muc2 mucin (green) show a few bacteria on the villi tip and between the villi, but no bacteria close to the crypt openings. (E) Duodenal villus tip. (F) Jejunal villus tip. (G) Ileal villus tip. (H) Fluorescent beads penetrate the mucus in ileum, experiment as in Fig. 1EF. Bar in A = 0.5 mm, bar in D1 = 100 µm, bars in D2, D3 and H = 50 µm and bars in E, F, G = 10 µm. Fig. 3. The domes of Peyer´s patches are covered by a penetrable mucus layer. (A) Mucus visualized with charcoal (arrow) on ileal explants containing three Peyer´s patches. Nonattached mucus demonstrated in the movie (supplemental video 6). (B) Mucus on domes penetrable to fluorescent beads as shown in Fig. 1EF. The epithelium (blue) of the dome is convex (viewed slightly tilted) and completely covered by beads. Bar in A = 0.5 mm, bar in B = 50 µm. Fig. 4. The stomach has mucus layers that are firmly attached to the epithelium and penetrable to fluorescent beads. (A) Stomach explants from the corpus, mucus visualized with charcoal 22 were gently aspirated (supplemental video 7, initial frame shown in A, left). A second movie recorded to illustrate the firm attachment of the outer layer in the stomach (supplemental video 8, initial frame is shown in A, right). (B) Mucus thickness of stomach corpus explants measured in paired experiments (n = 5, open circles) or with stimulated with carbachol and PGE2 (10 µM each; closed circles; arrow). Initial mucus thickness measured, mucus was removed and remaining mucus thickness measured (both time 0). Mucus was significantly thicker in explants after stimulation compared to unstimulated samples, P = 0.0048 (*). (C) Aspiration of mucus on stomach explants after stimulation of secretion by carbachol and PGE2 mucus (n = 5). (D) Karnovsky fixed, epoxy resin embedded sections stained with PAS (left) revealing a thin, striated mucus layer on top of the epithelium (arrow, bar = 10 µm). (E) Carnoy fixed paraffin sections stained with anti-Muc5ac (green) and DNA stain (blue) show Muc5ac positive goblet cells and mucus The epithelial surface imarked with dotted line (bar = 50 µm). (F) Fluorescent beads sedimented for 60 min into the mucus as described in Fig. 1EF: (1) unstimulated tissue; (2) tissue stimulated for 20 min with carbachol and PGE2 (10 µM of each); (3) mucus secreted after 10 min stimulation into an apical pH 3 buffer. Bars = 50 µm. 23 SUPPLEMENTAL MATERIAL Supplemental video 1. An explant from the proximal colon was mounted in the horizontal Ussing-type chamber, the mucus was gently aspirated using a Gilson Pipetman® while a movie was acquired with a digital camera attached to the stereomicroscope to illustrate mucus properties. After the first aspiration, charcoal was added once more to show that the inner mucus layer was still present. Supplemental video 2. An explant from the distal colon was mounted in the horizontal Ussing-type chamber, the mucus was gently aspirated using a Gilson Pipetman® while a movie was acquired with a digital camera attached to the stereomicroscope to illustrate mucus attachment. After the first aspiration, charcoal was added once more to show that the inner mucus layer was still present. Supplemental video 3. A mouse duodenal explant was mounted in the horizontal Ussingtype chamber, the mucus was gently aspirated using a Gilson Pipetman® while a movie was acquired with a digital camera attached to the stereomicroscope. Note the presence of fat and food particles in the mucus. After the first aspiration, charcoal was added once more to show that the charcoal sedimented down also between villi. Supplemental video 4. A mouse jejunal explant was mounted in the horizontal Ussing-type chamber, the mucus was gently aspirated using a Gilson Pipetman® while a movie was acquired with a digital camera attached to the stereomicroscope. Note the presence of food 24 particles in the mucus. After the first aspiration, charcoal was added once more to show that the charcoal sedimented down also between villi. Supplemental video 5. A mouse ileal explant was mounted in the horizontal Ussing-type chamber, the mucus was gently aspirated using a Gilson Pipetman® while a movie was acquired with a digital camera attached to the stereomicroscope. Mucus in the ileum is easily aspirated. After the first aspiration, charcoal was added once more to show that the charcoal sedimented down also between villi. Supplemental video 6. A mouse ileal explant containing a Peyer´s patch with four domes was mounted in the horizontal Ussing-type chamber, the mucus was gently aspirated using a Gilson Pipetman® while a movie was acquired with a digital camera attached to the stereomicroscope. The initial mucus on top of the domes is easily aspirated. After the first aspiration, charcoal was added once more to show that the charcoal sedimented down on the epithelium. Supplemental video 7. A mouse stomach explant was mounted in the horizontal Ussing-type chamber, the mucus was gently aspirated using a Gilson Pipetman® while a movie was acquired with a digital camera attached to the stereomicroscope. Note that mucus is mostly just moved around and not removed when only gentle aspiration is used. After the aspiration, charcoal was added once more to show that only little mucus was removed. Supplemental video 8. A mouse stomach explant was mounted in the horizontal Ussing-type chamber, the mucus was aspirated using a Gilson Pipetman® while a movie was acquired with a digital camera attached to the stereomicroscope. Substantial force must be applied to 25 remove any mucus in the stomach. After the aspiration, charcoal was added once more to show that after the outer mucus layer was removed, the inner mucus layer was still present.