Within the innate domain, pattern recognition receptors (PRRs) such as TLRs and cytoplasmic nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) play an essential role in mucin synthesis (58). TLRs are a family of 11 evolutionarily conserved transmembrane receptors which are located on the cell surface or on intracellular endosomes. These receptors are activated by pathogen-associated molecular patterns (PAMPs). This activation terminates with the induction of the NF-κB family of transcription factors and the upregulation of the immune response (25, 59).

Expression profiles of TLRs differ throughout the GI tract. Recently, by utilizing five strains of TLR reporter mice, the spatial expression of TLRs in intestinal epithelial tissue was visualized. TLR2, 4, 5, 7, and 9 were found to be minimally or not expressed in the small intestinal epithelium while TLR2, 4, and 5 expression levels were substantially higher in the colon (60). Further, TLR3 was expressed at similar levels in both small intestinal and colonic epithelial cells (60). Only TLR5 was expressed by goblet cells in the small intestine while colonic goblet cells were found to express TLR1, 2, 4, and 5 (61) suggesting intrinsic TLR-meditated mucin regulation in these cells.

Through their activation of TLRs, ligands such as lipopolysaccharide (LPS) found on the outer membrane of most Gram-negative bacteria, lipoteichoic acid (LTA) on the cell wall of Gram-positive bacteria, and flagellin found in bacterial flagella are all potent activators of MUC2 expression (25, 62). For instance, upon stimulation by their respective ligands TLR2/1, TLR4, and TLR5, promote the downstream activation of the NLRP6 inflammasome in subpopulations of sentinel goblet cells located at the entrance of colonic crypts. This inflammasome, a multiprotein complex located in the cytoplasm of these cells, functions as a sensor for cellular stresses and plays a key role in intestinal barrier maintenance, infection defense and mucosal renewal (54, 63). These TLR-initiated cascades stimulate the compound exocytosis of MUC2 and trigger mucin secretion from adjacent goblet cells through intercellular gap junction signals. This subsequent increased MUC2 secretion can thus aid in the expulsion of bacteria from the upper part of the crypts (54). Recent studies have shown that TLR4 activation is important in goblet cell response during Citrobacter rodentium infection (64) and can regulate the differentiation of goblet cells in intestinal organoids (65). The activation of TLR4, involving the binding of lipid A moiety of LPS to the LPS binding protein (LBP), can upregulate the expression of MUC2 through the Ras-MEK1/2-Erk1/2 and NF-κB pathways (66). Establishing whether the particular crypt location of the goblet cells is a determining factor in mucin production in response to various TLR ligands will be a worthwhile direction for future research.

Further evidence gleaned from genetic knockout models have helped highlight the differential effects of TLRs on mucin regulation. For instance, naïve Tlr1^−/− mice have defective production and/or secretion of MUC2 in the colon leading to a patchy and significantly depleted mucus layer (67). Tlr5^−/− mice display a mosaic phenotype; a subset of these mice develop spontaneous colitis while the majority do not. Interestingly, compared to wild-type mice, colitic Tlr5^−/− mice do not display the normal dual-layer of colonic mucus; only a disorganized and largely penetrable layer is present. In contrast, non-colitic Tlr5^−/− mice had a normal though slightly thinner mucus layer (68).

Scant research exists involving the interaction between TLRs, goblet cell function, and mucin regulation in intestinal parasitic infection in vivo and this area remains largely unexplored. In vitro, however, antigens from the intestinal trematode Gymnophalloides seoi have been found to induce the expression of both TLR2 and MUC2 in HT-29 cells in an IFNγ-dependent manner. Further, co-stimulation with G. seoi antigen and antibodies against both TLR2 and TLR4 have been shown to diminish MUC2 expression in HT-29 cells compared to those cells treated with the antigen only. Thus, the authors of this study hypothesize that the induction of MUC2 expression as an antiparasitic response in human IECs, may, at least in part, be a result of TLR activation (69). Additional in vivo and in vitro research will provide valuable insights into the interaction between TLRs, goblet cell function and mucin regulation in parasitic infection.

In contrast to the transmembrane TLRs, NLRs are a family of innate intracellular receptors (70). However, similar to TLR signaling, activation of NLRs such as NOD1 and NOD2 by intracellular ligands (i.e., bacterial peptidoglycans) ultimately results in the activation of important transcription factors, such as NF-κB, to induce immune responses (71). An enteric infection model using the helminth, Trichuris muris, has revealed that NOD1 and NOD2 receptors are necessary for MUC2 synthesis and parasitic expulsion; knocking out both of these genes (Nod-DKO) result in infected mice with lower goblet cell numbers and decreased MUC2 expression (72).

Other receptors can also impact mucin production via the innate response. LTA from Gram-positive bacteria can upregulate MUC2 expression by acting on platelet-activating factor receptor (PAFR) and, through a multistep process, activate the Ras-MEK1/2-Erk1/2 and NF-κB pathways (25). Moreover, flagellin signals through the glycolipid receptor asialoGM1 (ASGM1) ultimately lead, again, to upregulated expression of MUC2 (73). ASGM1-mediated upregulation of MUC2 involves the sequential activation of phospholipase C, an increase in calcium ion levels as well as ERK1/2 and NF-κB activation (73).

In the local draining lymph node, antigen presenting cells (APCs) such as dendritic cells and macrophages present the phagocytosed and processed antigen to aid in the differentiation of naïve CD4⁺ T cells to Th2 cells. These cells then secrete effector cytokines such as IL-13 and, thusly, contribute to mucus production and goblet cell hyperplasia (74). In addition, macrophages, primed by the canonical type 2 cytokines IL-4 and IL-13, can transition into alternatively activated or M2 macrophages (75). Moreover, alternatively activated macrophages can be generated by IL-33 and polarization was associated with increased induction of IL-13 (76), suggesting these APCs may also play a more direct role in the regulation of mucin via this cytokine.

ILCs are a recently discovered group of innate immune cells that play an essential role in host immunity, tissue protection, and adaptive immune regulation, particularly within the intestinal mucosal barrier (77–79). ILCs bridge the gap between the innate and adaptive immune responses by producing immune-regulatory cytokines. Based on the effector cytokines that ILCs secrete, the transcription factors that regulate their development, and markers dotting their cell surfaces, this family of cells can be subdivided into three groups: ILC1, ILC2, and ILC3 (77, 78).

Unlike the innate immune system which relies on germ-line encoded PRRs, the adaptive immune system generates specific receptors to recognize the substantial diversity of harmful antigens through a process called somatic recombination (93). The principal cell types of the adaptive immune system are T and B lymphocytes which are vital in maintaining gut homeostasis as well as host protection in GI diseases (94). Consequently, T lymphocytes play an important role in the regulation of mucin release by goblet cells (95). Initial studies demonstrated that during N. brasiliensis infection, anti-CD4 antibody treatment in mice prevented spontaneous recovery. These mice also displayed T-helper cell depletion along with a reduction in mucin levels, despite unchanged goblet cell counts (96).

The immune response to intestinal helminth infection is characterized as a Th2-dominant response accompanied by the upregulation of cytokines, such as interleukin (IL)-4, IL-5, and IL-13 (97, 98). Animal models of enteric infections including N. brasiliensis (99), Strongyloides ratti (100), T. muris (98), and Trichinella spiralis (101) have shown that these helminth infections are also accompanied by goblet cell hyperplasia and an increase in mucin production and secretion that aid in worm expulsion (102). It is postulated that these changes in mucin production and goblet cell hyperplasia are at least partially the result of the Th2-mediated immune response (103). As an activator of Th2 immune responses, signal transducer and activator of transcription factor 6 (Stat6) has been identified as a critical inducer of goblet cell hyperplasia (3, 104). This factor's crucial role is neatly illustrated in Hymenolepis diminuta infection; STAT-6 knockout mice infected with this tapeworm were unable to clear this infection due, in part, to diminished goblet cell response, unlike their IL-13 and IL-4 deficient counterparts at 12 days post-infection (105). Interestingly, it has been shown that goblet cell hyperplasia may also occur in a Th2-independent manner without the influence of IL-4 and IL-13 in some parasitic infections, including Schistosoma mansoni (106). It should also be noted that with the discovery of ILC2s, our understanding of the type 2 immune response has expanded. Both ILC2 and Th2 cells can be activated by IL-33, IL-25, and TSLP, and release type 2 effector cytokines (e.g., IL-5 and IL-13) during parasite infections, and thus, contribute to type 2 immunopathology. Further, Th2 cells respond to antigen stimulation by dendritic cells which also receive signals from cytokines released by ILC2s (107). These findings suggest that Th2 immune responses may be initiated by ILC2 since the activation of ILC2s occurs during the early phase of type 2 immune responses (108, 109).

Moreover, emerging evidence suggests a novel role for other cytokines in mucus production. Previously considered a non-factor in mediating parasitic expulsion, IL-22, a member of the IL-10 family of cytokines, has been shown to induce goblet cell hyperplasia and mucin release. IL-22-deficient (Il22^−/−) mice have defective goblet cell responses during N. brasiliensis infection despite strong Th2 cytokine induction (110). Furthermore, evidence suggests that, following infection with T. trichiura, the human gut accumulates IL-22-producing Th cells within the intestinal mucosa and the resultant increase in IL-22 production and Th2 cytokines promotes goblet cell hyperplasia and mucus production (111). Similarly, humans infected with Necator americanus, a parasitic hookworm, showed upregulated IL-22 production as well as a robust systemic and mucosal Th2 and T-regulatory (T_reg) response, ultimately promoting goblet cell hyperplasia and worm expulsion (112). In addition to quantitative changes, cytokines can also regulate the quality and composition of mucins. A recent study found that administration of IL-10 reduces endoplasmic reticulum (ER) stress and prevents the misfolding of MUC2 in an in vitro model (LS174T) of intestinal goblet cells (113). Consequently, T lymphocytes play an essential role in controlling the release of mucins through signaling by Th2 cytokines (e.g., IL-4, IL-5, and IL-13) and other mediators such as IL-22.

In addition to Th2 cytokines, several Th1 cytokines have been shown to regulate mucin biosynthesis. In the intestinal cancer cell line, LS180, it was observed that the pro-inflammatory cytokines, including IL-1, IL-6, and TNF-α, increased the expression of MUC2 mRNA (95). Due to the influx of Th1 cytokines, reduced mucin glycosylation via incomplete processing of N-glycans was also present (95). In addition, several in vitro and in vivo models have demonstrated the relationship between downregulated MUC2 expression and increased IL-6 in the context of colon cancer. Interestingly, this inverse relationship tends to be associated with liver metastasis and the promotion of tumor growth in mice (114, 115). Further, in BALB/c duodenal explants, macrophage-derived IL-1 and human rIL-1β have the ability to induce mucin secretion from goblet cells (116). The authors suggest that this dose-dependent process may act as a protective mechanism by aiding in the clearance of toxic substances from the gut in periods of mucosal inflammation (116). In contrast, in a murine model of C. rodentium infection, the Th1 cytokines, IFN-γ, and TNF-α, decreased intestinal mucin production and its speed of transport from the Golgi to secretory vesicles. Lending further support to this finding, in vitro treatment of infected and non-infected intestinal mucosal surfaces with IFN-γ and TNF-α decreased the number of goblet cells, mucus thickness and transport (117). The contrasting data in the above paragraph implies that the effect of Th1 cytokines on mucin synthesis, transport, and secretion depends critically on, not only, the type of cytokine but also the pathological process in question.

Though extensive, the presented results highlight the need for further exploration of mucin's diverse immunological functions and the impact that the immune system itself has on mucin production. Indeed, these findings establish the crucial role mucin plays in barrier and immune function as well as the importance of immune-associated signaling on the regulation of mucin composition and function. Figure 2 highlights various immunological regulations that affect mucin production and goblet cell function within the gut.

It is increasingly apparent that the enteric nervous system (ENS) also contributes to mucin production. Recent studies have shown that mucosal neurons, which are situated in close proximity to lamina propria immune cells (e.g., APCs and ILCs), interact with epithelial cells and act as key regulators of mucin production at intestinal mucosal surfaces. Acetylcholine (ACh) is a primary parasympathetic neurotransmitter that is released by preganglionic nerve fibers and the vagus nerve (118). Previously, it has been established that ACh is important for goblet cell degranulation and can induce mucin secretion (54, 119, 120).

Muscarinic ACh receptors (MRs) are one of the two types of cholinergic receptors along with nicotinic ACh receptors (NRs) (121). Of increasing interest, type 3 muscarinic receptors (M3Rs) have received significant attention for their key role in maintaining mucosal barrier function (122) and in regulating mucin production and secretion within the GI tract (123). In addition, M3Rs contribute to host defense against N. brasiliensis (124, 125) and C. rodentium (121). Despite N. brasiliensis infection, mice deficient in the muscarinic acetylcholine receptor M3 (Chrm3^−/−) show an absence of typical goblet cell expansion (125). These mice also display impaired immunity to C. rodentium along with decreased goblet cell number and MUC2 gene expression in the colon compared with WT mice on day 13 post-infection (121).

Similar to MRs, vasoactive intestinal peptide (VIP) also participates in regulating intestinal goblet cell numbers and function at baseline; VIP-deficient mice show impaired goblet cell development and reduced expression of MUC2 (126, 127). VIP is a 28-amino-acid peptide secreted by enteric neurons and regulates gut motility (126). There are three known receptors for VIP: the higher affinity receptors, VIP receptor 1 (VPAC1) and VPAC2, and the lower affinity receptor PAC1 (128). Among these, VPAC1 mRNA is the most highly expressed on the apical membrane of the intestinal epithelium and receives signals from VIP fibers (129). Although VPACs are expressed on airways (130) and ocular mucosa (131), there is no direct evidence that intestinal goblet cells express VIP receptors. However, a recent study has reported that ex vivo treatment with VPAC antagonists resulted in a substantial decrease in goblet cell counts in the mouse ileum indicating potential ongoing VIP regulation of goblet cell production (126). Furthermore, lamina propria ILC2 function is also regulated by VIP via VPAC2 during parasite infection (132).

Interestingly, recent studies have also indicated indirect regulation of mucin production by a neuropeptide called neuromedin U (NmU) via ILC2 (89, 133, 134). During N. brasiliensis infection, glial cells sense intestinal epithelial cell-derived IL-33 and N. brasiliensis excretory/secretory products (ESPs) and trigger NmU production in a MyD88-dependent pathway (133). NmU not only induces smooth muscle contractions but also binds to NmU receptor 1 (Nmur1) on ILC2s promoting the secretion of IL-13, and, potentially impacting mucin secretion (135).

Inflammatory bowel disease (IBD) is an umbrella term which includes chronic inflammatory conditions of the gastrointestinal tract such as ulcerative colitis (UC) and Crohn's disease (CD). UC is characterized by superficial inflammation radiating proximally from the rectum, whereas CD is characterized by zones of deeply inflamed and non-inflamed tissue that can extend throughout the GI tract (136). These conditions affect millions worldwide, and the incidence is increasing globally (137).

Early studies suggested that single nucleotide polymorphisms in certain mucin genes including MUC3A, MUC3B, MUC12, and MUC17, predisposed individuals to CD and UC, however, these findings have not held up in more recent genome-wide association studies (GWAS) (138). Meta-analyses of several GWAS have more recently identified the gene encoding MUC1 (139) and the locus of leucine-rich repeat kinase 2 (LRRK2) which contains the MUC19 gene, (140) to have significant associations with CD.

Both CD and UC are accompanied by dysregulation of mucin synthesis and altered post-translational modification leading to barrier dysfunction (138). In UC, changes including reduced glycosylation and sulphation (141) as well as increased sialylation alter the efficacy of the mucins present and hinder their ability to maintain effective intestinal barrier function (20) particularly with regards to bacterial penetration (142, 143). Healthy colonic mucus is often heavily sulphated with increasing levels of sulphation extending from proximal to distal regions and conferring increasing resistance to bacterial enzymatic degradation. In both CD and UC, however, this phenomena is muted (20, 141). In inflamed tissue, goblet cell depletion is present in both UC and CD compared with controls (144) and altered mucus layer thickness has also been found in both conditions (145).

UC is associated with a relatively thin and discontinuous mucus layer, goblet cell depletion and reduced MUC2 synthesis (145–147). Reduced sulfate content of MUC2 has also been noted, though compensatory and preferential secretion of this mucin in active disease results in overall unaltered sulfate levels in the colon of UC patients (147). Interestingly, MUC5AC, a mucin not normally present in the colon, has been found in UC patients undergoing surgery (148). In addition, site-specific increases in MUC1 expression and decreases in MUC2 expression were observed in UC patients at the site of crypt abscesses and adjacent to ulceration, respectively (149). Reduction in MUC9 (150) and MUC20 (151) gene expression was also noted in both active and quiescent UC compared with healthy controls. Increased gene expression of MUC16 has also been described in UC patients with active disease as well as those in remission compared to healthy controls (151). Sialylation also plays a similar role to sulphation, increasing the resistance to enzymatic degradation. In rectal biopsies of UC patients an increased average extent of sialylation per mucin oligosaccharide was noted rather than a simple increase in oligosaccharide chains (152). Intriguingly, glycan “profiles” have been established whereby healthy controls and those UC patients with inactive disease displayed similar glycan/glycosylation patterns; abundant levels of complex and larger glycans and relatively small amounts of shorter glycans. In opposition, those with active UC displayed increased presence of shorter glycans and a marked decrease in several complex glycans (153). Furthermore, these aberrant glycosylation profiles were associated with the degree of inflammation and severity of disease (153). Similarly, in a large-scale proteomics study, van der Post et al. established a core colonic mucus proteome, a set of 29 core secreted and transmembrane proteins that form the mucus barrier in healthy controls and UC patients in remission. Several of these proteins were found to be reduced in active UC patients including core structural components, MUC2 and IgGFc-binding protein (FCGBP), and other goblet cell products including calcium-activated chloride channel regulator 1 (CLCA1) and zymogen granule protein 16 (ZG16). Interestingly, this trend occurred independent of local inflammation and was associated with increased bacteria penetrability and activation of IL-18 (143). In active UC, contributing to the thinner and penetrable mucus layer, insufficient replenishment or exhaustion of sentinel goblet cells in response to successive microbial challenges has been noted and may precede the activation of disease and/or local inflammation (142, 143).

While the relationship between UC and mucus thickness is relatively well-established, the relationship regarding CD and mucus thickness remain under dispute. Early findings suggested CD patients displayed increased mucus layer thickness compared to healthy controls (146), however, more recent work has indicated that mucus layer thickness in CD patients was not significantly different compared with healthy controls (145). In seeming contradiction, a recent systematic review and meta-analysis concluded that, on average, patients with CD have a 34% reduction in total mucin levels due to significantly decreased levels of MUC5AC, MUC5B, and MUC7 (154). Similar to UC, ileal CD has also been linked with aberrant protein expression of MUC5AC and decreased expression of MUC2 (155). Aberrant expression of MUC6 has also been noted in ileal CD (156). Moreover, in the ileum of CD patients, a marked decrease in MUC1 mRNA was also observed in inflamed vs. non-inflamed tissue from the same patient (156). Interestingly, reduced gene expression of the transmembrane mucins, MUC3 and MUC4, as well as the secreted mucin MUC5B have been noted in non-inflamed tissue sections of CD patients compared with healthy controls (156) suggesting local inflammation is not necessarily a precursor for these alterations. Alterations in oligosaccharide length have also been noted in CD patients and although it has been established that levels of sulphation remain unchanged compared with healthy controls, changes in glycosylation levels in CD patients have not been thoroughly explored (20, 141, 147, 152).

From the above evidence, it is clear that alterations in mucin expression and function play a unique role in IBD. However, because of significant dispute in the literature, it is challenging to characterize distinct mucin expression profiles associated with IBD, and it should be noted that significant heterogeneity exists.

Colorectal cancer is the third most common cancer in the world and accounts for substantial mortality every year (157). The expression of secreted and transmembrane mucins is altered in colorectal cancer patients. MUC1 expression was found to be increased in colon cancer patients and was correlated with poor prognosis and metastasis (158). Though MUC1 is usually undetectable with tandem repeat peptide antibodies in healthy colons due to heavy glycosylation, this post-translational modification is reduced in colorectal cancer patients which may account for the observed differences (159, 160). In addition, MUC2 expression was found to be decreased in colorectal cancer instances, other than in mucinous adenocarcinomas (12, 161). Moreover, MUC5AC, a normal component of gastric mucus which is usually absent from the colon, was shown to be expressed de novo in colorectal cancer (162, 163).

Since the 1990s, the dextran sulfate sodium (DSS) model has been used extensively in rodents to understand the pathophysiology of colitis (164). The cytokine profile associated with this chemical model is predominantly within the Th1 immune response with the upregulation of the cytokines, IL-12, IFN-γ, and TNF-α (165). Animal studies using DSS have highlighted the important and diverse roles of mucins in modulating intestinal inflammation.

As previously mentioned, MUC2 is a secreted gel-forming mucin and the main structural component of intestinal mucus which contributes significantly to host protection in the context of intestinal inflammation. In vivo results show that rats placed on DSS regimen undergo goblet cell depletion; however, depending on the observed location within the colon, the expression of MUC2 was unaltered or increased (166). Under a different DSS regimen, mucin expression was shown to vary with the progression of colitis. Compared with controls, DSS-treated rats showed initial upregulation of MUC2 and MUC3, followed by a rapid reduction in expression over time (167). Within 12 h of DSS administration, decreased mucus thickness and increased mucus permeability in the colon allows commensal microbes to penetrate the inner mucus layer and reach the intestinal epithelial cells. It is thought that this early bacterial invasion plays a critical role in eliciting the infiltration of immune cells and driving the development of colitis (168).

Despite the lack of consistent expression patterns in the above results, it is evident that MUC2 is an important component of the protective mucus layer of the intestine. In fact, MUC2-deficient (Muc2^−/−) mice develop spontaneous colonic inflammation characterized by weight loss, changes in stool consistency, and increased expression of pro-inflammatory cytokines (169). Muc2^−/− mice also show increased severity of inflammation and disease activity when exposed to DSS (169). Therefore, it is clear that MUC2 offers significant protection against colonic inflammation and that a well-maintained colonic mucus layer is vital in preventing murine colitis. In contrast to this distinct protective role of MUC2, not all mucins have shown to have protective functions in a DSS-induced colitis model.

Unlike MUC2, MUC4 is a transmembrane mucin and a component of the glycocalyx found in the intestine. Mice deficient in MUC4 show resistance to DSS colitis and have reduced levels of pro-inflammatory cytokines compared to wild-type animals (170). Although the mechanism behind the protective role of MUC4 deletion is unknown, it is possible that Muc4^−/− mice respond by upregulating protective mucins in a compensatory manner to resist DSS-induced colitis. In fact, Muc4^−/− mice challenged with DSS colitis have been found to have higher expression of both MUC2 and MUC3 compared to wild-type mice (170). Similarly, mice deficient in MUC1, another transmembrane mucin, develop less severe colitis accompanied by a reduction in T cell infiltration (171). The lower severity of colitis in Muc1^−/− mice may also be attributed to compensatory increases in the expression of MUC2 and MUC3 (171, 172).

Besides chemical models, genetically modified animals are also commonly utilized to study intestinal inflammation and mucin production. One such model is the IL-10 knockout murine model (173). If kept in non-germ-free housing, mice deficient in IL-10 spontaneously develop colitis. This unprompted inflammation is accompanied by a reduction in the number of mucin-producing goblet cells in the intestine (173). In contrast, mice lacking the IL-1rn gene, coding for the IL-1 receptor antagonist, spontaneously developed IBD-like abnormalities including increased immune cell infiltration and secretion of pro-inflammatory cytokines, and had an increased number of goblet cells in the jejunum and ileum compared to wild-type mice. Dosh et al. reasoned that this rise in goblet cell number was due to increased expression of the transcription factors, Hath1 and Kruppel-like factor 4 (KLF4) in the inflammatory process (174). The contradictory findings of goblet cell number in IL-10 and IL-1rn deficient mice demonstrate the implications and importance of studying various genetic models reflective of the pathogenesis of intestinal inflammation. Furthermore, genetically altered mice with tamoxifen-induced villin-Cre-dependent intestinal deletion of kindlin 1 and 2 manifest UC-like features due to modified mucus composition and hydrophobicity. These mutant mice develop mucosal colonic inflammation secondary to defective tight junction morphology and extended paracellular space in the mucosal barrier. Here, defective tight junctions prevent the paracellular transport of phosphatidylcholine (PC) causing reduced mucus PC content and a >50% reduction in mucus hydrophobicity. Consequently, the mucosa was predisposed to microbial invasion and subsequent inflammation (175). This finding highlights the role of altered mucus composition as a driving event in UC and not merely a secondary result of inflammation.

In addition to immune markers, recently, significant emphasis has been placed on studying the role of autophagy in mediating intestinal inflammation, and genetically modified animal models have been created to study this phenomenon. Autophagy is an evolutionarily conserved, catabolic cellular mechanism whereby cytoplasmic contents are delivered to, and degraded in, the lysosome (176, 177). A link between autophagy and intestinal inflammation has been proposed, and genome-wide association studies have identified the gene encoding ATG16L1, an autophagy-related protein, as a susceptibility locus for CD (178). Further, mice deficient in the autophagy-related protein, ATG7 in the intestinal epithelial cells (Atg7^ΔIEC), have been shown to develop more severe symptoms of colitis when placed on a DSS regimen compared to wild-type mice (179). These mice also had reduced expression of anti-microbial and anti-parasitic peptides, and an increased abundance of gut microbial content. Moreover, evidence suggests that Atg7^ΔIEC mice also have diminished release of intestinal mucins, particularly MUC2, and a less thick mucus layer. These findings indicate that the altered abundance, as well as the increased severity of colitis in Atg7^ΔIEC mice, could at least be partially caused by the reduced levels of intestinal mucins released from goblet cells (179).

Lack of proper glycosylation has been illustrated in UC patients (20, 141, 180) and several genetic models regarding this process provide insight into the implications of altered glycosylation in intestinal inflammation. As mentioned previously, O-linked oligosaccharides, in particular core 1- and core 3- derived mucin type O-glycans, are crucial components in the maintenance and stability of the colonic mucus layer, helping to prevent the penetration of this layer by microbial species via protease degradation and to avert unwarranted activation of the immune response (28, 29, 181). Found throughout the colon, core 1 β1,3-galactosyltransferase (C1GalT1) controls the synthesis of core 1 O-glycans (181). Due to the impaired glycosylation and disrupted mucus integrity found when knocking out this protein's corresponding gene, mice with intestinal epithelium-specific deficiency of core 1 derived O-glycans (C1galt1^ΔIEC) develop spontaneous colitis in the distal regions of the colon (181) characterized by myeloid cell infiltration, crypt abscess, epithelial ulceration, goblet cell loss, reduced mucin levels, disrupted mucus layer, and increased epithelial–microbial interaction (153, 180). In contrast to C1GalT1, core 3 β1,3-N-acetylglucosaminyltransferase (C3GnT) expression, which regulates core 3 O-glycan formation, is more localized to the proximal colon (181). C3Gnt^−/− mice show increased susceptibility to colitis and colorectal cancer (182–184). Loss of both intestinal core 1- and 3-derived O-glycans generate mice that develop colitis ranging from the proximal to distal regions of the colon and display earlier onset and more severe intestinal inflammation when compared to C1galt1^ΔIEC mice and C3Gnt^−/− mice suggesting the loss of these vital components confers compounding deleterious effects (182, 184). Further, the above phenomena with regards to impaired glycosylation of the mucins and its effects on intestinal inflammation seem to be dependent, at least somewhat, on the presence of the resident microbiota; several studies have shown that antibiotic treatment lessens the severity of colitis and boosted mucus layer integrity in these models (180, 181, 183). Thus, evidence from the C1galt^ΔIEC, C3Gnt^−/−, and double knockout models suggest proper glycosylation is crucial in maintaining the integrity of the colonic mucus layers, and suggests these O-glycans are a key factor in protecting the underlying epithelium from abnormal microbial interaction and preventing unsolicited intestinal inflammation.

From the above evidence, it is clear that within different physiological environments, different mucins, and different alterations in those mucins can greatly affect the host susceptibility to intestinal inflammation. These findings highlight the essential role of mucin in the pathogenesis of intestinal inflammation and exemplify the importance of maintaining mucin levels within a healthy homeostatic range. Because of this complexity, further research must be carried out to clearly elucidate the various roles of mucins in IBD.

Trichuris trichiura is a soil-transmitted helminth which affects millions of people, particularly children, around the world (185). To gain a more comprehensive understanding of the immune response against T. trichiura, enteric parasitic animal models have been developed. One such model which has been extensively used in both our laboratory and others is the murine infection, T. muris. Resistant mice or those that are able to expel worms successfully display a characteristic Th2 immune response with the upregulation of IL-4 and IL-13. In contrast, susceptible hosts such as AKR mice develop a Th1 immune response associated with chronic infection and increased worm burden (186). Changes in mucin production, both qualitative and quantitative, can affect host protection against parasitic infections, and thus, the protective role of mucins in T. muris infection is increasingly being explored.

There are several methods through which mucins contribute to parasitic clearance. Firstly, it has been shown that some mucins, such as MUC5AC, have direct damaging effects on worms and reduce their viability (31). Thus, it comes as no surprise that mice deficient in MUC2 or MUC5AC show delayed worm expulsion in response to T. muris infection (31, 187). Secondly, mucins can create a thick and impermeable physical barrier to protect the underlying epithelial cells from T. muris invasion. In fact, T. muris infection leads to an increase in the thickness of glycocalyx, particularly due to the upregulation of MUC4, MUC13, and MUC17 proteins (103). Interestingly, the serine proteases secreted by T. muris can degrade MUC2, but not MUC5AC, giving this mucin considerable influence on the successful clearance of these invaders (31, 188).

Entamoeba histolytica, a human protozoan parasite, causes amebic colitis, and liver abscess, a condition collectively called amebiasis (189). Mostly present in the developing world, symptoms manifest in 10% of infected individuals possibly due to variation in host immune response (189). Interestingly, MUC2 plays a vital role in the development and progression of amebiasis (189). After ingestion of contaminated food or water, E. histolytica colonizes the colonic outer mucus layer by binding to glycans on the MUC2 molecule via its Gal/GalNAc-lectin (190). The protozoa then cleave MUC2 by glycosidases and proteases and subsequently, comes in to contact with intestinal epithelial cells (191, 192). Thus, the strength of the mucin layer is a key player in the physical defense against E. histolytica-induced inflammation and epithelial invasion. In addition to MUC2 degradation, altered MUC2 production is found in intestinal amebiasis. E. histolytica has been found to bind to αvβ3 integrins on goblet cells and stimulate the hypersecretion of mucus by exocytosis (193). Contrasting data suggests, however, that E. histolytica impairs the regulation of Math1 transcription factor required for goblet cell differentiation and can actually lead to decreased mucus production (194). In addition, during E. histolytica infection, MUC2, possibly by acting either as a cAMP ligand or by activating TLRs, can promote elevated levels of antimicrobial peptides such as cathelicidins (195).

Clostridium difficile is an anaerobic, spore-forming bacterium that causes worldwide epidemics with significant mortality rates (196). Initial infection by this enteric pathogen is characterized by an acute inflammatory response with a substantial influx of neutrophils, diarrhea, and weight loss (197). Fortunately, murine colitis models utilizing C. difficile have provided a better understanding of this infection. Previously, it has been shown that patients with C. difficile infection secreted acidic mucus primarily composed of MUC1 and have decreased MUC2 expression indicating defective mucosal barrier function (198). Moreover, patients with C. difficile infection exhibited altered mucus composition with higher GLcNAc and galactose levels, but lower GalNAc levels (198). Intriguingly, fecal microbiota transplantation (FMT) has received a growing interest as an effective therapeutic strategy to treat this infection. Recently, it has been shown that upon IL-33 treatment, C. difficile-infected mice exhibited increased goblet cell number and mucin production via activation of IL-13-secreting ILC2s and FMT rescued IL-33 expression in the colon after antibiotic-mediated depletion (199). Other enteric bacterial infections have also illustrated the implications of alteration within the mucus layer. For instance, animal studies have shown that deficiency in the transmembrane mucin, MUC1, results in increased susceptibility and more severe intestinal damage in response to infection with Campylobacter jejuni (200). Similarly, mice deficient in MUC2, the main ingredient of colonic mucus, had increased intestinal permeability and were more susceptible to S. enterica serovar Typhimurium infection (201, 202).

In addition to alteration in quantity, post-translational modifications can also affect the ability of mucin to maintain intestinal barrier integrity. One such modification, the addition of fatty acids called palmitoylation, is particularly important for maintaining this barrier function. Inactivation of the enzyme, fatty acid synthase (FAS) in intestinal epithelial cells prevents palmitoylation of MUC2, and ultimately leads to an increase in intestinal permeability (203, 204). Improper sulfonation, or the addition of sulfate anions to mucins, has similar effects of intestinal permeability in C. jejuni infection (205). In addition, barrier function and pathogen adhesion are largely regulated by the diverse glycosylation pattern of mucins. Analysis of gastric mucin glycosylation profiles reveal important roles of terminal α1,2-fucose residues on Lewis-b and H type 1 structures expressed on MUC5AC (206). By utilizing α1,2-fucosyltransferase-deficient (FUT2^−/−) mice, it has been established that the binding capacity of the infectious agent, Helicobacter pylori, to Lewis-b and H-type antigens is impaired due to the loss of gastric MUC5AC fucosylation (207). Similarly, norovirus infection is dependent on the fucosylation status of soluble mucins within the GI tract (206, 208). It is speculated that reduced mucin fucosylation in the GI tract results in decreased mucus thickness and increased intestinal permeability to pathogens (206). Further, inhibition of the enzyme, core 3 β1,3-N-acetylglucosaminyltransferase (C3GnT) which plays a crucial role in the addition of O-linked oligosaccharides, can alter the structural and functional features of mucins and has been shown to decrease MUC2 levels and increase intestinal permeability (184, 209). Moreover, goblet cells in mice resistant to chronic T. muris infection contain high levels of sulphated mucins in contrast with the goblet cells of susceptible mice which are dominated by sialylated mucins. Consequently, sulphation of mucins promoted by Th2 immune mediators, such as IL-13, augments the protection offered by mucins by aiding in their ability to resist degradation (210).

Table 1 summarizes the various and dynamic changes in mucins and the mucus layer in the context of the aforementioned pathologies and animal models.