Composition of the Two Mucus Layers.

We next investigated the components of the two colonic mucus layers by separating reduced and alkylated samples of mucus by composite agarose-PAGE (AgPAGE). Upon gel staining with Alcian blue to visualize mucin bands, we detected two major bands for Muc2, corresponding to Muc2 monomer (the smallest detectable component of Muc2, marked M) and a likely dimer (3) that were similarly represented in the loose- and firm-mucus layer preparations (Fig. 1C). Mass spectrometry of the tryptic peptides showed Muc2 as the only gel-forming mucin detected in these bands. The identified peptides encompassed 30% of the protein (excluding the PTS domains) and included most of the peptides expected not to be glycosylated [Fig. 1A and supporting information (SI) Table S1]. Only minor peptide differences, localized to the N-terminal part, were observed between Muc2 from the firm and loose mucus. The intensity of the bands and peptide representation suggest that the Muc2 mucin is a major constituent of both the firm and loose mucus layers. Upon analysis of the small-sized protein components of the loose and firm mucus by PAGE and Coomassie blue staining, identical patterns for the two mucus layers were observed (Fig. S1). A detailed comparison identified proteins that were intracellular components, serum proteins, and likely mucus constituents. Out of these, the secreted proteins and proteins with large extracellular domains as well as their association to the loose and/or firm layers is presented in Table 1 and Tables S2 and S3, revealing that the proteins were present in both the firm and loose mucus layers (some proteins were only identified under less-stringent conditions). The expression of some of these proteins was further verified by immunostaining (Fig. S2) showing Clca3 expression in the granules of the goblet cells as shown before (6), a localization also shown for Fcgbp (7). That the composition of the loose and firm mucus layers is almost identical suggest that the loose mucus layer is generated from the firm mucus layer.

When the amount of the Muc2 mucin recovered from an identical sealed surface area was compared, the firm layer was estimated to contain at least the double amount of Muc2 as compared with the loose layer when measured by the band intensity of Alcian blue stained gels (data not shown). Considering that the loose mucus layer is approximately twice as thick as the firm (Fig. 1B), this suggests a higher concentration of Muc2 by a factor of four in the firm relative to the loose mucus layer.

Properties of the Two Mucus Layers.

The Muc2 mucin from the intestine has been observed to be partly insoluble in chaotropic salts like guanidinium chloride (8, 9). To study whether the loose and firm mucus layers had different properties in this respect, both layers extracted with guanidinium chloride were separated by centrifugation into a soluble supernatant and an insoluble pellet. A clearly visible pellet could be detected only in the firm (F) layer, but not in the loose (L) (Fig. 2A). After reduction, alkylation, and dialysis, the samples were analyzed for mucins by AgPAGE and Alcian blue staining (Fig. 2B). The pellet of the guanidinium-insoluble firm mucus layer (Fp) contained the Muc2 mucin but only small amounts in the loose pellet.

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Fig. 2.

The firm and loose mucus layers have different properties. (A) Loose (L) and firm (F) mucus were extracted by 6 M guanidinium chloride into soluble and insoluble pellet fractions. The insoluble pellet was photographed and is clearly visible in the firm mucus. (B) The soluble (s) and insoluble (p) fractions of loose (L) and firm (F) mucus after reduction of disulfide bonds were analyzed for mucins by AgPAGE and the gel stained with Alcian blue. The firm mucus pellet (Fp) contained Muc2 mucin, whereas the loose pellet (Lp) was almost devoid of Muc2. (C) The mucus thickness measured in rat distal colon in vivo overlaid with protease inhibitors (n = 4) or control (n = 16). The protease inhibitors (PI) were added after the first removal of the loose mucus. The firm (F) mucus is presented together with the regeneration of the loose (F+) after 20, 40, and 60 min and a final measurement of the firm (F) mucus after removal of the loose mucus. Values are mean ± SEM. *, P < 0.05 vs. untreated. The germ-free (GF) mice had a loose (white) and firm (gray) mucus layer as shown to the right (n = 5). (D) Mucus from the same measured surface area of the loose material was removed at 60 min after protease inhibitor (PI) treatment, reduced and analyzed on AgPAGE, and stained with Alcian blue. M, Muc2 monomer. (E) Western blot analysis of reduced loose and firm mucus after 60 min with or without protease inhibitor treatment. The bands were detected by the anti-MUC2N3 (anti-N3) or anti-MUC2C3 (anti-C3) antisera. (F) Equal amounts by volume of guanidinium chloride-insoluble mucus pellets from mouse colon (A) were treated or nontreated with trypsin for 3 h, resulting in volume expansion of the pellet in the trypsin-treated sample as shown by the photo and a graphic representation of the pellets.

Because the loose mucus layer had different properties than the firm layer, yet the Muc2 mucin was a major component, we hypothesized that proteolytic cleavages could be responsible for the transformation from firm to loose mucus layer. After removal of the loose mucus in rat colon, a protease inhibitor (PI) mixture inhibiting serine and cysteine proteases was added to the firm mucus layer in vivo (Fig. 2C). This caused a decreased replenishing rate of the loose mucus (F + 20 and 40 min), but the thickness of the firm layer was not affected (F 60 min). To our surprise, the newly formed loose mucus as analyzed by reduced AgPAGE revealed an increased amount of intact full-length Muc2 mucin in the PI-treated sample by a factor of three (Fig. 2D). The loose mucus was also analyzed for proteolytic fragments of the Muc2 mucin by reduced PAGE and Western blot using anti-Muc2 N- and C-terminal specific antisera (Fig. 2E). The band patterns were similar for the PI-treated loose layer and firm layer that showed identical patterns irrespectively of PI addition. However, the nontreated loose mucus was different, suggesting that the Muc2 mucin of the loose mucus layer normally undergoes additional proteolytic cleavages, deleting fragments especially from the C terminus. The smaller volume and higher Muc2 concentration in the PI-treated loose mucus imply a protease-dependent volume expansion in this layer. The presence of a loose mucus layer also in germ-free animals suggests that the formation of a loose mucus is due to endogenous processes/proteases (Fig. 2C Right). The identities of these activities are still not known, but all proteases identified by proteomics are presented in Table S4. As the mucus gel is still intact, some of the proteolytic cleavages must maintain an intact Muc2 polymer that allow volume expansion. Such phenomena are most likely due to cleavages in the disulfide bond-stabilized, compact N- and C-terminal domains (4, 10). This interpretation is substantiated by the observation that treatment of the guanidinium chloride-insoluble Muc2 pellet with trypsin does not dissolve the pellet, but allows a volume expansion (Fig. 2F).

Bacteria Are Excluded from the Inner Stratified Mucus Layer.

Immunostaining of Carnoy-fixed mouse distal colon with an anti-Muc2 antiserum (green) identified two major mucus layers in addition to Muc2-positive goblet cells (Fig. 3A and control Fig. S3). The staining of the inner layer was characterized by a well organized stratified lamellar appearance, suggesting that it was formed by sheets of polymerized Muc2 (s in Fig. 3 A and C). This is consistent with the notion that the N-terminal trimers in the Muc2 polymers (4) should favor such an organization. This layer was after fixation ≈25-μm thick and probably represented the firm mucus layer observed in vivo (Fig. 1B). Under this, a thin and more intensely stained layer was observed that could correspond to a not yet fully organized mucus layer (a in Fig. 3 A and B). This is directly linked to the goblet cells with Muc2-positive streaks (white arrow). As the firm mucus layer is attached to the epithelia in colon, it is possible that this anchoring is to the goblet cells and mediated by these Muc2-containing streaks. The outer Muc2 mucus layer (o in Fig. 3A) is characterized by a differently stained Muc2 and probably corresponds to the loose mucus layer. The border was relatively sharp, suggesting a controlled transition from the inner stratified to the outer layer.

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Fig. 3.

The firm mucus layer is devoid of bacteria. (A) Muc2-positive goblet cells and overlaying mucus layers in a section of the mouse distal colon were detected by using the anti-MUC2C3 antiserum (green). The section is counterstained with DAPI to visualize nuclei. An inner stratified mucus layer (s) is linked via Muc2-stained threads (white arrow) to the goblet cells of the surface epithelia. The lowest part of the inner mucus layer is stained differently (a), suggesting a different organized form compared to the stratified (s) layer. The outer mucus layer (o) is mixed with the luminal content. (Scale bar: 50 μm.) (B) Magnification of the interface between the epithelial surface and the mucus layer, marked as in A. (C) Magnification of the inner mucus layer displayed a stratified pattern suggesting a lamellar organization. (D) Combined Muc2 immunostaining (green) as in A and FISH analysis using the general bacterial probe EUB338-Alexa Fluor 555 (red) of distal colon shows Muc2-positive goblet cells and an inner stratified (s) mucus layer on the epithelium. This layer is devoid of bacteria, which can only be detected in the outer mucus layer. The inner mucus generates a spatial separation between the cells and the microflora. (Scale bar: 20 μm.) (E) Semiquantitative PCR of bacterial 16S gene using DNA isolated from equal surface areas of the loose (L) and firm (F) mucus demonstrated more bacteria in the loose mucus layer. Amplification was done for 20, 25, and 30 cycles as indicated.

Next, we analyzed the tissue sections for bacterial presence by in situ hybridization using a general 16S rRNA probe (Fig. 3D, negative control in Fig. S4). Bacteria were detected (red) in the outer mucus layer and were excluded from the inner stratified layer (s). This observation was supported by semiquantitative PCR using primers specific for conserved regions of the bacterial 16S rRNA genes performed on DNA isolated from the loose and firm mucus layers. Considerably less bacterial DNA was detected by PCR in the firm mucus layer (Fig. 3E) where the smaller amounts of bacteria in the firm layer could be due to difficulties in fully removing the loose layer. Thus the inner firm mucus layer, devoid of bacteria, could act as a physical barrier impenetrable to bacteria, whereas the expanded Muc2 in the loose mucus layer is a major habitat for commensal bacteria.

Measurements of the Mucus Thickness in Vivo.

The animals were anesthetized and prepared and the mucus measured with a micropipette connected to a micromanipulator in a 30° angle as described before for rat (5) and adapted to mouse (17). The mucus thickness, the vertical distance between the cell surface and the luminal mucus surface, was calculated (18). The results are expressed as mean ± SEM. To compare single values, Student's t test for paired or unpaired data was used. The differences were regarded as significant at P < 0.05. After removal of the loose layer during mucus measurements in rat, the firm mucus was covered with 2× complete EDTA-free protease inhibitor (Roche) in PBS. Mucus measurements were performed at specified times, followed by a second removal. Untreated rats were used as controls by following the same protocol.

SDS–Agarose Composite Gel Electrophoresis for Separation of Mucins.

Mucus from the colon was removed from an identical, measured epithelial surface by suction (loosely adherent) or scraped (firmly adherent), and protease inhibitors and complete EDTA-free protease inhibitor (Roche) were added. The samples, equalized to identical surface area were reduced in sample buffer with 100 mM dithiotreitol DTT at 95°C and alkylated by iodoacetamide or 4-vinyl pyridine (2.5 molar excess of DTT). A composite gel (AgPAGE) containing agarose (0.5–1% gradient), acrylamide (0–6%), and glycerol (0–10%) was used for analysis (19). The ImageJ software (National Institutes of Health) was used for relative quantification of Alcian blue stained bands.

Polyacrylamide Gel Separation and Western Blot Analysis.

Loose and firm mucus was sampled from the measured area or from the distal half of a dissected colon, with fecal pellets removed, and reduced by DTT in sample buffer and analyzed by 4–12% SDS/PAGE. The gels were stained by Coomassie with Imperial stain (Pierce) or blotted by semidry Western blot to Immobilone P membranes (Millipore). Muc2 was detected with a polyclonal serum against the human MUC2 C-terminal peptide PHYVTFDGLYYSYQGNC (anti-MUC2C3) reacting with the mouse Muc2 or with a serum against the N-terminal D3 domain (20). The HRP-conjugated secondary antibody was detected by Supersignal West Pico (Pierce).

Trypsin Digestion of Proteins, LC-MS/MS Analysis, and Protein/Mucin Identification.

Alcian blue-stained bands from AgPAGE or whole lane (total protein content in 22 bands) from 4–12% SDS/PAGE stained with Imperial stain (Pierce) were selected and excised. The proteins were digested in-gel by trypsin (Promega) and the peptides eluted and analyzed by nano-LC coupled to a hybrid linear ion trap-FT-ICR MS equipped with a 7T ICR magnet (LTQ-ICR(FT); Thermo) (21). Searches were performed by using MASCOT (Matrix Science). The search parameters were set to MS accuracy 5 ppm, MS/MS accuracy 0.5 Da, one missed cleavage allowed, fixed carbamidomethyl, S-pyridylethyl or propionamid modification of cysteine, and variable modification of oxidized methionine. The mass spectra from the LC-ICR MS/MS experiments were searched against an in-house mucin database containing the assembled mouse mucin sequences (www.medkem.gu.se/mucinbiology/) or the nonredundant protein sequence database downloaded from the National Center for Biotechnology Information (NCBI) October 11, 2007. The mucin bands in each lane from AgPAGE were merged, and the results from three analyses were assembled. The total protein content from one SDS/PAGE lane were merged (22 excised bands), and three separate analysis of loose and firm were compared.

Histology and Immunostaining.

Segments of the distal colon without washing from 10- to 14-week-old animals were fixed in Methanol–Carnoy's fixative. Paraffin-embedded sections were dewaxed and hydrated. Antigens were retrieved by Retrievagen A (BD). Sections were stained with Alcian blue/PAS or by the anti-MUC2C3 antiserum with FITC-conjugated goat anti-rabbit immunoglobulins (DAKO) and DAPI. Pictures were obtained by using a Nikon Eclipse E1000 fluorescence microscope, and a LSM 510 (Zeiss) or a Radiance 2000 (Bio-Rad) confocal microscope.

Isolation and Preparation of Soluble and Insoluble Mucins.

Loose and firm mucus samples were extracted in 6 M guanidinium chloride (22) supplemented with protease inhibitors 2× Complete protease inhibitor mix (Roche), stirred overnight at +4°C and centrifuged for 20 min at 16,000 × g. Extractions were repeated three times. Pellets were photographed, and samples were reduced in guanidinium chloride with DTT at 37°C for 3 h and alkylated with iodoacetamide overnight. The samples were dialyzed against water and solubilized in sample buffer.

Trypsin Treatment of Insoluble Mucus Pellet.

Guanidinium chloride extracted mucus was washed three times in 100 mM Tris·HCl (pH 8.5) and mixed with 10 μg of trypsin (Lonza) or buffer as control. The mucus was pelleted by centrifugation at 16,000 × g for 20 min and incubated at 37°C for 3h. The volumes of the pellets were photographed with bromophenol blue in the supernatant.

PCR of Bacteria 16S rRNA Genes in Loose and Firm Mucus.

The loose mucus was sampled from the distal part of a dissected colon, and the colon was washed once with PBS before sampling the firm mucus, both loose and firm were from an identical surface area. DNA was prepared with the Nuclospin tissue kit (Macharey Nagel) with a preceding digestion with 2 mg of Lysozyme. Samples were analyzed by PCR with universal primers directed against a region of the 16S rRNA gene common to most bacteria: set1 (forward 5′-CCATGAAGTCGGAATCGCTAG-3′and reverse, 5′-ACTCCCATGGTGTGACGG-3′) (bp 1302–1394 in bacteria EU622773) and set2 (forward, 5′-TCCTACGGGAGGCAGCAGT-3′; reverse, 5′-GGACTACCAGGGTATCTAATCCTGTT-3) (bp 339–780 in bacteria EU622773) (23). The PCR (95°C 30 s, 58°C 30 s, 72°C 45 s) was performed for 20, 25, or 30 cycles, with an initial step of 5 min at 95°C and final extension of 5 min at 72°C by using TaqDNA polymerase (NEB). Products were analyzed by agarose gel electrophoresis. Both PCR sets show similar results represented by set 2 in Fig. 3E.

We acknowledge the Proteomics Core Facility and Centre for Cellular Imaging at University of Gothenburg. This work was supported by Swedish Research Council Grants 7461, 8646, and 20680 and the Swedish Research Council provided equipment. Support was also provided by the Swedish Cancer Foundation, the Swedish Foundation for Strategic Research–Mucosal Immunobiology and Vaccine Center, and the IngaBritt and Arne Lundberg Foundation.