Mucin Gene Deficiency in Mice Impairs Host Resistance to an Enteric Parasitic Infection

1The mucus barrier is an essential part of the innate immune system which hydrates and protects the underlying epithelia. The gel-like properties of the barrier are mainly due to the polymeric mucins that are the main secretory products of epithelial goblet cells. The colonic epithelium expresses mainly MUC2/Muc2 in large amounts which is stored in bulky apical granules of the goblet cells and is the most important factor determining the goblet cell morphology. Muc2 forms a heterogeneous mucus barrier that is proposed to contain 2 distinct layers; a “loose” outer layer that bacteria can penetrate and an adherent inner layer that excludes bacteria from direct contact with the underlying epithelia.

2Alterations or absence of MUC2 production can lead to many common human disorders such as colon carcinoma, ulcerative colitis, and celiac disease. A role for Muc2 in the suppression of colorectal carcinoma has also been suggested because Muc2 knockout (KO) mice spontaneously develop colitis and adenomas that progress to invasive adenocarcinoma, suggesting an important function for this mucin in colonic protection. Furthermore, missense mutations in the Muc2 gene results in aberrant Muc2 oligomerization, leading to endoplasmic reticulum stress and subsequently increased susceptibility to colitis.

3Hyperplasia of mucin-producing goblet cells has been described in a number of parasitic infections, including Nippostrongylus brasiliensis, Hymenolepis diminuta, Trichinella spiralis, and Trichuris muris. Putative mechanisms underlying the protective role of mucins against infectious agents include the demonstration of trapping of Hymenolepis diminuta and Trichinella spiralis in the mucus and inhibition of parasite motility and feeding capacity. Goblet cell response, in all 4 of these nematode models, is thought to be under the control of a T helper (TH) 2-type immune response and is considered as a potential effector mechanism. A number of goblet cell bioactive factors such as resistin-like molecule-β (Relm-β), intelectin, and calcium-activated chloride channel-3 have been suggested to play an important role in nematode infection. However, a definitive and precise role of mucins, the main secreted product of goblet cells, in host defense in intestinal nematode infection remains to be elucidated.

4The nematode T muris inhabits the cecum of mice and is closely related at the morphologic, physiologic, and antigenic levels to Trichuris trichuria, the causative agent of chronic trichuriasis in human beings. In this parasitic infection, strains resistant to chronic infection (BALB/c, C57BL/6) expel the parasites through the generation of a TH2-type immune response, whereas susceptible strains (AKR), which do not expel the worms, develop a TH1-type immune response. In this study, we demonstrated that the increase in Muc2, the main determinant of mucus barrier properties, correlates with worm expulsion. In the absence of Muc2 there is a delay in worm expulsion, but interestingly Muc5ac is up-regulated in the Muc2-deficient mice before expulsion. Moreover, Muc5ac is up-regulated in the wild-type (WT) mice that are resistant to infection, but not in those unable to expel. The physical properties of the mucus barrier are also altered during infection, resulting in a less-porous network, with overall changes having a direct effect on the viability of the whipworm. Collectively, these data show for the first time a protective role for mucins in nematode infection.

5Breeding pairs of Muc2-KO mice originally produced by gene mutation and their WT (C57BL/6) littermates (Albert Einstein Medical College, New York, NY) were kept at the animal facilities of McMaster University (Hamilton, ON, Canada). AKR, BALB/c (Harlan, UK), and severe combined immunodeficient (SCID) mice were maintained in the Biological Services Unit at Manchester University. The protocols used were in accordance with guidelines by the McMaster University Animal Care Committee, Canadian Council on the Use of Laboratory Animals, and the Home Office Scientific Procedures Act (1986). All mice were kept in sterilized, filter-topped cages, and fed autoclaved food in the animal facilities. Only 6- to 10-week-old male mice were used.

6The techniques used for T muris maintenance and infection were described previously. Mice were orally infected with approximately 100–300 eggs for a high-dose infection and <15 eggs for a low-dose infection. Worm burdens were assessed by counting the number of worms present in the cecum as described previously.

7A 1-cm segment or the whole cecum (rolled) was fixed in 10% neutral buffered formalin or 95% ethanol and processed with the use of standard histologic techniques. Sections were treated with 0.1 mol/L KOH for 30 minutes before staining with periodic acid Schiff (PAS) reaction. Slides were counterstained with either H&E or 1% fast-green. Standard immunohistochemical and immunofluorescent staining methods were used to determine the levels of Muc2, Muc5ac, Relm-β, and trefoil factor 3 (Tff3).

8Immunodetection was carried out with the use of a polyclonal antibody raised against a murine Muc2 (mMuc2). Commercially available 45M1 antibody was used for the detection of mouse Muc5ac. The mouse Muc5b-specific antibody was a kind gift from Dr Camille Ehre (University of North Carolina, Chapel Hill). Commercially available mRelm-β (Abcam, Cambridge, UK) and mITF (Santa Cruz Biotechnology Inc, Santa Cruz, CA) antibodies were used to detect Relm-β and Tff3, respectively. Detection of bromodeoxyuridine (BrdU) incorporated into nuclei was carried out with the use of a monoclonal anti-BrdU antibody (AbD Serotec, Oxford, UK).

9The cecum was gently flushed with phosphate-buffered saline and scraped, and mucus was solubilized in 8 mol/L guanidium chloride. Subsequently, extracted mucus samples were reduced with 50 mmol/L dithiothreitol and carboxylmethylated with 0.125 mol/L iodoacetamide before electrophoresis on a 1% (wt/vol) agarose gel. Mucins were detected after Western blotting with mucin-specific antisera.

10Cecal tissue isolated from BALB/c and AKR mice was cut longitudinally, washed with phosphate-buffered saline, and kept hydrated in a 6-well plate. Blue fluorescently labeled polymer microspheres (0.1 μm; Dukes Scientific, Dorchester, United Kingdom) were placed on top of the luminal surface of the cecum (set as a reference) and their position was analyzed with the use of the Nikon (Melville, NY) C1 Upright confocal microscope. Three-dimensional optical stacks were taken every 5 μm and combined to obtain a z-stack at the time points stated.

11The CellTiter-Glo luminescent cell viability assay (Promega, Madison, WI) was carried out according to manufacturer's instructions. Relative light units were calculated per worm as follows: relative light unit = (sample light units − blank light units)/number of worms. Substrate only was used as a blank control, whereas worms were boiled before homogenization for negative controls. To determine recovery of energy status, worms recovered were washed extensively in Dulbecco's modified Eagle's medium, added to 6-well plates with LS174T cells (maintained as previously described by Hayes et al) for 24 hours before measuring adenosine triphosphate (ATP) levels.

12All results are expressed as the mean ± standard error of the mean. Statistical analysis was performed with the use of SPSS Version 16.0 (SPSS Inc, Chicago, IL). Statistical significance of different groups was assessed with parametric tests (one-way analysis of variance with post test after statistical standards or paired Student t test). P <.05 was considered statistically significant.

13It has been well documented that susceptible (AKR) mice harbor the T muris worms until patency (day 35 after infection; Figure 1A), whereas the resistant (BALB/c) mice start expelling worms by day 14 after infection, and expulsion is achieved by day 21 after infection (Figure 1A). Changes in the production of Muc2, the main gel-forming constituent of intestinal mucus, were explored within the cecum of AKR or BALB/c mice exposed to a high-dose T muris infection. Immunohistochemical staining and reverse transcription–polymerase chain reaction (RT-PCR) analysis for Muc2 (Figure 1B and C) showed that significantly higher amounts of Muc2 were expressed within the cecal crypts of the resistant mice on day 21 after infection than in the naïve and susceptible mice; a similar staining pattern was observed with the PAS reagent (Supplementary Figure 1). This increase in goblet cell number and Muc2 levels was restricted to the niche of the parasite, was not observed in the colon (Supplementary Figure 1), and correlated with worm expulsion. Therefore, to further understand the role of Muc2 in T muris infection, we performed a high-dose infection in Muc2-deficient mice on the resistant C57BL/6 background.

14A high-dose T muris infection established in both WT and Muc2-KO mice showed no marked difference in the number of worms at day 13 after infection (Figure 2A). However, as infection progressed, there was a significant decrease in worm burden in the WT mice, evident by day 15 after infection (46% reduction) and with a 84% decrease over establishment levels by day 20 after infection. In contrast, in the Muc2-deficient mice there was no decrease in worm burdens until after day 20 after infection, although mice did eventually expel their parasites.

15We next sought to determine whether the delay in worm expulsion in the KO mice was due to an alteration to the adaptive immune response to T muris infection. Interleukin-4 (IL-4) and interferon-γ levels in intestinal tissue were not detectable in the naïve, WT, and KO mice. Furthermore, there was no significant difference in IL-4 or interferon-γ levels in intestinal tissues between both strains on day 20 after infection (Figure 2B). Consistent with the local immune response, there was no significant difference in IL-4 and IL-13 production from in vitro concanavalin A–stimulated spleen cells (Figure 2B). Thus, despite the delay in worm expulsion, Muc2 deficiency had no significant effect on generation of the TH2-type immune response in T muris infection. The crypt architecture, an indicator of inflammation, changed during infection; there was an increase in crypt length on day 15 after infection in WT and KO mice which was more pronounced in the KO mice (Figure 2C). With the use of the highest position of BrdU-positive cells in the crypts as a measure of rate of epithelial cell turnover, it was clear that cell turnover was higher in the naïve KO mice than in the WT mice. However, there was no significant difference in epithelial cell turnover between the KO and WT mice on day 20 after infection (Figure 2D), indicating that the delay in worm expulsion in KO mice was not associated with an alteration of the “epithelial escalator.”

16Despite the similar number of goblet cells (as defined by Relm-β and Tff3; Supplementary Figure 2) in the infected and noninfected KO and WT mice, there was a significant difference between the number of PAS-positive goblet cells (Figure 3A). As with the resistant BALB/c mice, there was a significant increase in the numbers of PAS-positive goblet cells in WT mice after infection. Although there was significant impairment in the development of hyperplastic goblet cells in the KO mice, unexpectedly by day 15 after infection, there was an increase in PAS-positive goblet cells, with significant elevation by day 30 after infection (Figure 3A; Supplementary Figure 3).

17After exposure to T muris the levels of Muc2 were significantly elevated in the WT mice (Supplementary Figure 3). As expected, no Muc2-positive goblet cells were seen in the KO mice. Similarly, higher amounts of Muc2 (assessed by Western blotting after agarose gel electrophoresis) were present in the content of mucus collected in the WT mice after infection (Figure 3B). Although there was little evidence of mature, glycosylated Muc2 in the KO mice, interestingly, on day 21 after infection, there was a faint band consistent with the electrophoretic migration of Muc2 in these mice (red box; Figure 3B). This, along with the PAS-positive goblet cells, suggested the presence of another polymeric mucin after infection. To identify this mucin, the mucus (pooled from 5 KO mice) was analyzed by Western blotting after agarose gel electrophoresis. A mouse Muc5b-antiserum did not show any bands (data not shown). In contrast, a Muc5ac monoclonal antibody identified bands in the mucus samples from infected mice (Figure 4A). Immunofluorescence microscopy (Figure 4C), RT-PCR (Figure 4D), and tandem mass spectrometry (data not shown) confirmed the de novo expression of Muc5ac after infection in the KO mice. Furthermore, the PAS-stained material after agarose gel electrophoresis showed coincidence with the Muc5ac reactive band (Figure 4B arrow), suggesting that Muc5ac is a significant component of the mucus in the KO animals. No marked changes were observed in the expression of the cell surface mucins, Muc1, Muc4, and Muc17, which are thought to contribute to mucosal protection (Supplementary Figure 3).

18Unexpectedly, Muc5ac expression was also significantly up-regulated in the WT mice on days 14 and 21 after infection (Figure 5). In contrast to the KO mice, Western blotting showed that Muc5ac mucin was not the main component in the mucus, because the main PAS bands migrated further than the broad, Muc5ac-reactive band (Figure 5arrow) and was coincident with Muc2 staining bands (data not shown). However, the de novo expression of Muc5ac was only observed in the resistant mouse models (high dose in C57BL/6 and BALB/c mice) and not in the susceptible models (low dose in BALB/c, high dose in AKR and SCID mice) (Figure 5C). Immunofluorescence microscopy and immunohistochemistry confirmed the expression of Muc5ac after infection (days 15 and 21) in the cecal crypts of the resistant models (Figure 5D). No reactivity was observed in the susceptible models (data not shown).

19Fluorescently labeled beads were used to investigate mucus permeability after infection (day 19) in BALB/c and AKR mice (Figure 6A). The beads traveled to a depth of approximately 100 μm over a 60-second period in both strains. Thereafter, there was a reduction in diffusion rate of the beads in the resistant (BALB/c) mice. However, the beads traveled significantly further in the mucus of susceptible (AKR) mice over a 20-minute period.

20ATP measurements were carried out to determine the energy status of worms in resistant and susceptible mice as a measure of worm vitality. As infection progressed (day 21 after infection) in the AKR mice, there was a significant increase in the ATP production by the worms (Figure 6B). In contrast, there was a marked reduction in ATP production in the worms isolated from the BALB/c mice. However, these worms were not irreversibly damaged because they recovered their ATP production when transferred to in vitro culture with the colonic LS174T cell line for 24 hours (Supplementary Figure 4). Importantly, worms taken from Muc2-deficient mice showed a comparable drop in energy status during worm expulsion (Supplementary Figure 4).

21It is well established that T muris survives by eliciting a TH1 response in mice susceptible to chronic infection in the absence of a TH2 response. In common with all other studies of intestinal helminth immunity, multiple effectors under immunologic (TH2) control are probably operating during worm expulsion. Although we already know that IL-13–mediated regulation of epithelial cell turnover and smooth muscle contractility can contribute to worm expulsion, little detail is known about the protective role of the secreted barrier, ie, mucus. Previously, we have shown that the TH2-type immune response in resistance plays an important role in the development of goblet cell hyperplasia. Some reports have also suggested that mucus produced from goblet cells has an important role in trapping and removing nematodes from the intestine. The polymeric mucins are responsible for the physical properties of the mucus barrier, and changes in mucins are associated with pathophysiology of a number of gastrointestinal disorders. It has also been shown that deficiency in the main component of the intestinal mucus barrier, Muc2, leads to an abnormal morphology of the colon and contributes to the onset and perpetuation of dextran sulfate sodium–induced experimental colitis.

22In this study we demonstrated, using the T muris model, that Muc2 increased in resistance (restricted to the cecum, the niche of the parasite) which correlated with worm expulsion. However, this was not the case for the mice susceptible (AKR) to T muris infection, supporting the hypothesis that Muc2 contributed to host protection in nematode infection. A distinct functional role for Muc2 in host protective immunity in T muris infection was shown in the Muc2-deficient mice. These animals exhibited a significant delay in worm expulsion even though the adaptive immune response was unaltered; similar TH2-type immune responses were shown in Muc2-deficient and WT control mice after infection. Unexpectedly, de novo expression of Muc5ac was observed just before worm expulsion in the Muc2 KO mice and resistant mouse models, but not in the susceptible models. Overall, the network properties of the intestinal mucus barrier are different between resistance and susceptibility, and the changes in the parasitic niche can have damaging effects on the vitality of the parasite. To our knowledge this is the first direct demonstration for a functionally protective role of gel-forming mucins in nematode infection.

23Analysis of cecal mucus from Muc2-deficient mice showed that Muc5ac was the only polymeric mucin present in the mucus after infection. Moreover, in WT mice, although not the main mucin (which is Muc2), for the first time in a nematode infection we show the up-regulation of Muc5ac after intestinal infection. Several studies have elucidated that TH2-type cytokines such as IL-13 have the ability to up-regulate MUC5AC/Muc5ac expression levels. Therefore, the up-regulation in Muc5ac expression observed after infection, in both WT and Muc2-deficient mice, may be a result of IL-13 production. Interestingly, this de novo expression of Muc5ac was observed in all the resistant models (TH2-type response) but in none of the susceptible models (TH1-type response) of T muris infection. Although this mucin is predominantly found in airway and stomach mucus, studies on patients with ulcerative colitis and adenocarcinomas have shown MUC5AC expression in the intestine along with MUC2. However, this is the first time that Muc5ac expression has been implicated in response to an enteric parasitic infection.

24We observed no discernible PAS-positive goblet cells throughout the cecum of Muc2-deficient mice without infection. However, this was not due to the absence of goblet cell lineage as the expression of Tff3, and Relm-β was observed in the cecum of both infected and noninfected WT and Muc2-deficient mice. This observation corroborates with the findings of Van der Sluis et al whereby the expression of Tff3 was observed, despite the lack of PAS-positive goblet cells. Muc2 seems to be the main phenotypic determinant of goblet cells, and, in the absence of Muc2, goblet cells lose their characteristic goblet-like shape and specific staining, but the goblet cell lineage is still present. Interestingly, after infection there was an increase in PAS-positive goblet cells in the Muc2-deficient mice. Although the size of the goblet cells in Muc2-deficient mice was smaller than in those in WT mice, their emergence correlated with worm expulsion.

25We have shown a functional role for the mucus barrier in host protective immunity to T muris infection because in the absence of Muc2, worm expulsion is significantly delayed. Moreover, the physical properties of the mucus barrier are changed after infection, although the details of how these changes contribute to protection remains to be fully elucidated. However, one possibility is that, in the susceptible mice, the lower levels of Muc2 result in a network that may compromise defense because of inappropriate presentation or concentration of other host defense proteins in the environment of the worms. Whereas, in the resistant mice, other proteins (such as Relm-β, Tff3, and angiogenin) may be retained and effectively concentrated at the sites of worm infection. This may be by specific interactions with Muc2 or with the infected induced Muc5ac, or by the physical constraints imposed by the mucin network, thus rendering the host interface unsuitable for worm reproduction and/or survival which results in expulsion. Indeed, changes in the niche of the parasite do have a detrimental effect on the parasite, because worms extracted from mice during worm expulsion clearly have a reduced energy status than worms extracted from the susceptible mice. This reduction in the worm vitality is reversible if worms are transferred to a “favorable” environment, supporting the notion that expulsion reflects damaged, but not killed parasites.

26Another explanation supported by our finding, which is by no means mutually exclusive, is that the physical nature of the mucus barrier is changed in such a way as to facilitate worm expulsion. We have shown that around the time of worm expulsion the mucus barrier is less porous in the resistant mice than in the susceptible mice, and this alteration in physical properties of the barrier after infection may directly affect the niche of the worms. The intestinal mucus barrier is proposed to comprise “loose” outer layer and a less porous, adherent inner layer. The results from the bead penetration assay showed that after 60 seconds the beads travelled to a depth of approximately 100 μm in the mucus from the susceptible and resistant mice, suggesting the properties of the loose layer are similar in both. However, the beads traveled at different rates thereafter, suggesting that the differences in network properties observed between the resistant and susceptible mice are mainly in the inner adherent layer of the barrier. This alteration may physically constrain the worms, thus affecting the niche.

27What might be the role of the infection-induced mucin Muc5ac in protection against the worms? Muc5ac is assembled in a different manner to Muc2 and does not possess the disulphide-resistant cross-links present in Muc2 and may result in a mucus gel with different rheologic properties. Indeed, Muc5ac is a main component of airway mucus, and, unlike the intestinal barrier which is normally an adherent Muc2-rich gel, a specific functional requirement in the airways makes a transportable mucus gel. Thus, Muc5ac may change the rheologic nature of the mucus gel and, in conjunction with the intestinal muscle hypercontractility (controlled by TH2 response), could physically aid worm expulsion. This is consistent with the observations of mucus trapping in N. brasiliensis and T. spiralis infection, in which globules of mucus trap worms, which are then transported out of the intestine. Another interesting possibility raised by the data is that expulsion occurs in 2 phases: an early phase influenced by Muc2 and a final, clearance phase that occurs independently of Muc2, possibly involving Muc5ac.

28In conclusion, this study clearly shows that the mucus barrier is a significant component of a well-coordinated response in the gut to worm expulsion. Even though T muris has an intracellular niche within the gut epithelium, in resistance as the “epithelial escalator” displaces worms, it may be that the overall changes in barrier have a subsequent significant detrimental effect on the worm itself, and the additional changes in the physical properties of mucus contribute to the efficient elimination of the worms from the intestinal lumen. Moreover, it further highlights the functionally dynamic and highly regulated nature of the mucus barrier during immunologically mediated intestinal disease.