Intestinal mucus barrier: a missing piece of the puzzle in food allergy

The sudden rise in food allergy over recent decades has led to various theories including the role of environmental factors in sensitization.Gastrointestinal (GI) mucins have recently been implicated in maintaining gut homeostasis: mice lacking a functional mucus layer (Muc2−/− mice) are more susceptible to colitis and GI infection, in addition to severe oral tolerance breakdown following sensitization with a food allergen. Thus, further research will be necessary to understand how functional changes in O-glycosylated mucins might contribute to the epidemic of food allergy.Factors including reduced consumption of dietary fiber and/or increased consumption of food additives are emerging as key determinants of altered mucus barrier function and mucin−gut microbiota interactions which appear to play an important role in regulating food allergy. The prevalence of food allergies has reached epidemic levels but the cause remains largely unknown. We discuss the clinical relevance of the gut mucosal barrier as a site for allergic sensitization to food. In this context, we focus on an important but overlooked part of the mucosal barrier in pathogenesis, the glycoprotein-rich mucus layer, and call attention to both beneficial and detrimental aspects of mucus–gut microbiome interactions. Studying the intricate links between the mucus barrier, the associated bacteria, and the mucosal immune system may advance our understanding of the mechanisms and inform prevention and treatment strategies in food allergy. The prevalence of food allergies has reached epidemic levels but the cause remains largely unknown. We discuss the clinical relevance of the gut mucosal barrier as a site for allergic sensitization to food. In this context, we focus on an important but overlooked part of the mucosal barrier in pathogenesis, the glycoprotein-rich mucus layer, and call attention to both beneficial and detrimental aspects of mucus–gut microbiome interactions. Studying the intricate links between the mucus barrier, the associated bacteria, and the mucosal immune system may advance our understanding of the mechanisms and inform prevention and treatment strategies in food allergy. Western countries have been experiencing rising rates of food allergies without a clearly identified cause [1.Gupta R.S. et al.Prevalence and severity of food allergies among US adults.JAMA Netw. Open. 2019; 2e185630Crossref PubMed Scopus (250) Google Scholar, 2.Lyons S.A. et al.Food allergy in adults: substantial variation in prevalence and causative foods across Europe.J. Allergy Clin. Immunol. Pract. 2019; 7: 1920-1928Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 3.Stephen-Victor E. et al.Dietary and microbial determinants in food allergy.Immunity. 2020; 53: 277-289Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar], and common food allergies include peanuts, treenuts, seafood, and cow's milk. Food allergy prevalences in Europe and among US adults were recently estimated at 6% and 10%, respectively [1.Gupta R.S. et al.Prevalence and severity of food allergies among US adults.JAMA Netw. Open. 2019; 2e185630Crossref PubMed Scopus (250) Google Scholar,2.Lyons S.A. et al.Food allergy in adults: substantial variation in prevalence and causative foods across Europe.J. Allergy Clin. Immunol. Pract. 2019; 7: 1920-1928Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar]. Food allergen sensitization (see Glossary) occurs when regulatory immune responses fail to keep up with the induction of allergen-specific proinflammatory immune responses [3.Stephen-Victor E. et al.Dietary and microbial determinants in food allergy.Immunity. 2020; 53: 277-289Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar]. This drastic increase in food allergy prevalence has led researchers and clinicians to propose that modern lifestyles and environmental changes are a trigger [3.Stephen-Victor E. et al.Dietary and microbial determinants in food allergy.Immunity. 2020; 53: 277-289Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar]. Identifying the underlying mechanisms that causually connect environmental factors to sensitization is an upcoming area of research that would inform treatment options and prevention strategies. Food allergies can manifest at all ages, and the large spectrum of symptoms and immune profiles range from immediate IgE-mediated responses to delayed non-IgE-mediated responses [4.Connors L. et al.Non-IgE-mediated food hypersensitivity.Allergy, Asthma Clin. Immunol. 2018; 14: 56Crossref PubMed Scopus (22) Google Scholar,5.Anvari S. et al.IgE-mediated food allergy.Clin. Rev. Allergy Immunol. 2019; 57: 244-260Crossref PubMed Scopus (42) Google Scholar]. Sensitization to allergens and the initiation of allergic disease begin at barrier sites which share key protective features such as a cohesive epithelium that overlies a dynamic immune system (Figure 1). Atopic march often refers to atopic dermatitis in the newborn, food allergies in childhood, and asthma that can persist as chronic rhinitis into adulthood [6.Zhu T.H. et al.Epithelial barrier dysfunctions in atopic dermatitis: a skin–gut–lung model linking microbiome alteration and immune dysregulation.Br. J. 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Nagler C.R. Influences on allergic mechanisms through gut, lung, and skin microbiome exposures.J. Clin. Invest. 2019; 129: 1483-1492Crossref PubMed Scopus (25) Google Scholar]. We focus here on a specific and understudied component of the mucosal barrier in the gut: the gastrointestinal (GI) mucus layer. Mucus is a crucial feature of mucosal surfaces, including the respiratory and digestive tracts (Figure 1) [10.Pelaseyed T. et al.The mucus and mucins of the goblet cells and enterocytes provide the first defense line of the gastrointestinal tract and interact with the immune system.Immunol. Rev. 2014; 260: 8-20Crossref PubMed Scopus (512) Google Scholar, 11.Martens E.C. et al.Interactions of commensal and pathogenic microorganisms with the intestinal mucosal barrier.Nat. Rev. Microbiol. 2018; 16: 457-470Crossref PubMed Scopus (164) Google Scholar, 12.Benam K.H. et al.Mucociliary defense: emerging cellular, molecular, and animal models.Ann. Am. Thorac. Soc. 2018; 15: S210-S215Crossref PubMed Scopus (9) Google Scholar]. The mucus layer in the airways plays a protective role similar to that of the digestive tract, and mucus regulation is impaired during disease state such as asthma, causing pathophysiology [13.Fahy J.V. Dickey B.F. Airway mucus function and dysfunction.N. Engl. J. Med. 2010; 363: 2233-2247Crossref PubMed Scopus (848) Google Scholar]. O-glycosylated mucin glycoproteins are the building blocks of mucus layers, and provide lubricant and filtering properties to help the expulsion of luminal contents and prevent potential pathogens from entering close contact with the epithelium, while allowing proper absorption of digested nutrients [10.Pelaseyed T. et al.The mucus and mucins of the goblet cells and enterocytes provide the first defense line of the gastrointestinal tract and interact with the immune system.Immunol. Rev. 2014; 260: 8-20Crossref PubMed Scopus (512) Google Scholar]. In addition to mucins, a diverse range of secreted immunological factors such as antimicrobial peptides and secretory immunoglobulin A (sIgA) provide potent weapons to prevent infections [14.Johansson M.E.V. Hansson G.C. Immunological aspects of intestinal mucus and mucins.Nat. Rev. Immunol. 2016; 16: 639-649Crossref PubMed Scopus (310) Google Scholar]. We discuss clinically important but unconnected links in the literature to provide clues about how the gut mucus barrier is important in maintaining oral tolerance and limiting exposure to immunologically active allergen residues. An important element that regulates the GI mucosal barrier is the gut microbiome, and this has recently been proposed as an important modulator of food allergies [15.Stefka A.T. et al.Commensal bacteria protect against food allergen sensitization.Proc. Natl. Acad. 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As the broader aspects of the gut microbiome links in sensitization have been reviewed elsewhere [3.Stephen-Victor E. et al.Dietary and microbial determinants in food allergy.Immunity. 2020; 53: 277-289Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar,9.Kemter A.M. Nagler C.R. Influences on allergic mechanisms through gut, lung, and skin microbiome exposures.J. Clin. Invest. 2019; 129: 1483-1492Crossref PubMed Scopus (25) Google Scholar,22.Wesemann D.R. Nagler C.R. The microbiome, timing, and barrier function in the context of allergic disease.Immunity. 2016; 44: 728-738Abstract Full Text Full Text PDF PubMed Google Scholar], and evidence is mounting about how the commensal microbiota impacts the integrity and maintenance of the mucus layer [23.Desai M.S. et al.A dietary fiber-deprived gut microbiota degrades the colonic mucus barrier and enhances pathogen susceptibility.Cell. 2016; 167: 1339-1353Abstract Full Text Full Text PDF PubMed Scopus (935) Google Scholar, 24.Schroeder B.O. et al.Bifidobacteria or fiber protects against diet-induced microbiota-mediated colonic mucus deterioration.Cell Host Microbe. 2018; 23: 27-40Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, 25.Earle K.A. et al.Quantitative imaging of gut microbiota spatial organization.Cell Host Microbe. 2015; 18: 478-488Abstract Full Text Full Text PDF PubMed Google Scholar], we focus our attention on the mucus–microbe interactions. We present a novel perspective on the functioning of the intestinal mucus layer, and we discuss both the beneficial and detrimental roles of mucus–microbe interactions in food allergy. Mucus layers are composed of highly O-glycosylated mucin glycoproteins that are secreted by goblet cells (GCs) and expand on the apical surface of the epithelium [26.Javitt G. et al.Assembly mechanism of mucin and von Willebrand factor polymers.Cell. 2020; 183: 717-729Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar]. This organized layer works as a barrier to exclude gut microbes while remaining permeable to essential nutrients and macromolecules [27.Ermund A. et al.Studies of mucus in mouse stomach, small intestine, and colon. I. Gastrointestinal mucus layers have different properties depending on location as well as over the Peyer's patches.Am. J. Physiol. Gastrointest. Liver Physiol. 2013; 305: G341-G347Crossref PubMed Scopus (197) Google Scholar] (Figure 2). Moreover, the structure and composition of the mucus layer varies along the length of the digestive tract in parallel with intestinal morphology and function [27.Ermund A. et al.Studies of mucus in mouse stomach, small intestine, and colon. I. Gastrointestinal mucus layers have different properties depending on location as well as over the Peyer's patches.Am. J. Physiol. Gastrointest. Liver Physiol. 2013; 305: G341-G347Crossref PubMed Scopus (197) Google Scholar]. In the stomach, mucin-5AC (MUC5AC) is the predominant gel-forming mucin and the mucus is structured into two thick layers – a firmly adherent layer attached to the epithelium overlain by a loose adherent layer [28.Atuma C. et al.The adherent gastrointestinal mucus gel layer: thickness and physical state in vivo.Am. J. Physiol. Gastrointest. Liver Physiol. 2001; 280: G922-G929Crossref PubMed Google Scholar]. These thick layers have been proposed to work as a buffer to facilitate proton secretion while protecting the gastric tissue from the acidic luminal content [29.Lewis O.L. et al.A physics-based model for maintenance of the pH gradient in the gastric mucus layer.Am. J. Physiol. Gastrointest. Liver Physiol. 2017; 313: G599-G612Crossref PubMed Scopus (12) Google Scholar]. In the small and large intestines, mucin-2 (MUC2) is the predominant secreted mucin, although mucus layers harbor different structures [27.Ermund A. et al.Studies of mucus in mouse stomach, small intestine, and colon. I. Gastrointestinal mucus layers have different properties depending on location as well as over the Peyer's patches.Am. J. Physiol. Gastrointest. Liver Physiol. 2013; 305: G341-G347Crossref PubMed Scopus (197) Google Scholar]. The mucus layer in the small intestine is a single loose and permeable layer that permits commensals to enter closer to epithelial cells such that these are further sampled by lamina propria antigen-presenting cells [30.Shan M. et al.Mucus enhances gut homeostasis and oral tolerance by delivering immunoregulatory signals.Science. 2013; 342: 447-453Crossref PubMed Scopus (342) Google Scholar]. By contrast, in the colon, two distinct layers of mucus have been described, the outer layer being more loose and permeable to bacteria whereas the inner one is more firmly attached to the epithelium and is impermeable to bacteria [28.Atuma C. et al.The adherent gastrointestinal mucus gel layer: thickness and physical state in vivo.Am. J. Physiol. Gastrointest. Liver Physiol. 2001; 280: G922-G929Crossref PubMed Google Scholar,31.Johansson M.E.V. et al.The two mucus layers of colon are organized by the MUC2 mucin, whereas the outer layer is a legislator of host-microbial interactions.Proc. Natl. 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In addition to the protein backbone of mucins, O-glycosylation is an important feature that contributes to the viscoelasticity of mucus, thus promoting its activity as a lubricant to help to expel particles, parasites [37.Bergstrom K. et al.Proximal colon-derived O-glycosylated mucus encapsulates and modulates the microbiota.Science. 2020; 370: 467-472Crossref PubMed Scopus (29) Google Scholar,38.Sharpe C. et al.A sticky end for gastrointestinal helminths; the role of the mucus barrier.Parasite Immunol. 2018; 40e12517Crossref PubMed Scopus (23) Google Scholar], and thus potential allergens. GI mucins are composed of four main types of O-glycan structural cores, in addition to terminal fucosylation, sialylation, and sulfation, and these post-translational modifications are differentially distributed along the digestive tract [11.Martens E.C. et al.Interactions of commensal and pathogenic microorganisms with the intestinal mucosal barrier.Nat. Rev. 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O-glycosylation contributes to mucin conformation and plays an essential role in protecting their protein backbone from proteolysis by microbial enzymes [40.Bergstrom K. et al.Core 1- and 3-derived O-glycans collectively maintain the colonic mucus barrier and protect against spontaneous colitis in mice.Mucosal Immunol. 2017; 10: 91-103Crossref PubMed Scopus (62) Google Scholar,41.Van Der Post S. et al.Site-specific O-glycosylation on the MUC2 mucin protein inhibits cleavage by the Porphyromonas gingivalis secreted cysteine protease (RgpB).J. Biol. Chem. 2013; 288: 14636-14646Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar]. Consistently, mice with defects in intestinal O-glycosylation display a disorganized and permeable colonic mucus layer and develop microbiota-dependent spontaneous colitis [40.Bergstrom K. et al.Core 1- and 3-derived O-glycans collectively maintain the colonic mucus barrier and protect against spontaneous colitis in mice.Mucosal Immunol. 2017; 10: 91-103Crossref PubMed Scopus (62) Google Scholar,42.Fu J. et al.Loss of intestinal core 1-derived O-glycans causes spontaneous colitis in mice.J. Clin. Invest. 2011; 121: 1657-1666Crossref PubMed Scopus (231) Google Scholar,43.Tobisawa Y. et al.Sulfation of colonic mucins by N-acetylglucosamine 6-O-sulfotransferase-2 and its protective function in experimental colitis in mice.J. Biol. Chem. 2010; 285: 6750-6760Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar]. Recent characterization of colonic MUC2+ cells by single-cell RNA-seq has identified specific subpopulations of GCs that differ based on gene expression profiles, localization in the epithelium, and mucus secretion patterns [44.Nyström E.E.L. et al.An intercrypt subpopulation of goblet cells is essential for colonic mucus barrier function.Science. 2021; 372eabb1590Crossref PubMed Scopus (10) Google Scholar]. At the surface of the epithelium, both crypt- and intercrypt-derived mucus generate a mucus layer that is impenetrable to bacteria-sized 1 μm beads [44.Nyström E.E.L. et al.An intercrypt subpopulation of goblet cells is essential for colonic mucus barrier function.Science. 2021; 372eabb1590Crossref PubMed Scopus (10) Google Scholar]. However, 0.2 μm beads and 2000 kDa fluorescein-labeled dextran, mimicking macromolecules or nutrients, could only penetrate the intercrypt mucus. Intriguingly, these populations could be distinguished by differential lectin staining, revealing different glycosylation patterns of the secreted mucins [44.Nyström E.E.L. et al.An intercrypt subpopulation of goblet cells is essential for colonic mucus barrier function.Science. 2021; 372eabb1590Crossref PubMed Scopus (10) Google Scholar]. This study paves the way towards a high-resolution understanding of the diverse roles of the mucus at the interface between the microbiota and the immune system. Mucin production is regulated by luminal microbial products as well as by immune factors [14.Johansson M.E.V. Hansson G.C. Immunological aspects of intestinal mucus and mucins.Nat. Rev. Immunol. 2016; 16: 639-649Crossref PubMed Scopus (310) Google Scholar]. As described in the lung, type 2 inflammation elicited by parasite infections was shown to induce intestinal GC hyperplasia and mucus secretion through the IL-13–IL-4Rα–STAT6 pathway [38.Sharpe C. et al.A sticky end for gastrointestinal helminths; the role of the mucus barrier.Parasite Immunol. 2018; 40e12517Crossref PubMed Scopus (23) Google Scholar,45.Oeser K. et al.Conditional IL-4/IL-13-deficient mice reveal a critical role of innate immune cells for protective immunity against gastrointestinal helminths.Mucosal Immunol. 2015; 8: 672-682Crossref PubMed Google Scholar]. In addition, cholinergic agonists, as well as histamine and prostaglandin E2 released by effector cells, are potent inducers of mucus secretion in the small and large intestines [46.Halm D.R. Halm S.T. Secretagogue response of goblet cells and columnar cells in human colonic crypts.Am. J. Phys. 2000; 278: C212-C233Crossref PubMed Google Scholar,47.Specian R.D. Neutra M.R. Mechanism of rapid mucus secretion in goblet cells stimulated by acetylcholine.J. Cell Biol. 1980; 85: 626-640Crossref PubMed Google Scholar]. Although similar type 2 immune responses are found in food allergy, little is known about the subsequent outcome for intestinal mucus production and composition. Mucus depletion has been reported in the distal colon of food-allergic patients with proctitis [48.Carroccio A. et al.Chronic constipation and food intolerance: a model of proctitis causing constipation.Scand. J. Gastroenterol. 2005; 40: 33-42Crossref PubMed Scopus (0) Google Scholar]. In addition, mucus replenishment was observed after allergen avoidance, suggesting allergen-driven depletion [48.Carroccio A. et al.Chronic constipation and food intolerance: a model of proctitis causing constipation.Scand. J. Gastroenterol. 2005; 40: 33-42Crossref PubMed Scopus (0) Google Scholar]. This contrasts with allergic asthma, where IL-13-driven overproduction of mucus in the airways is a pathological outcome [49.Lambrecht B.N. et al.The cytokines of asthma.Immunity. 2019; 50: 975-991Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar]. Colonic mucus depletion has also been described in active ulcerative colitis (UC, a type of inflammatory bowel disease, IBD) where sentinel GCs at the entrance of the crypts do not produce mucus in response to bacterial stimuli, in contrast to GC in healthy controls or in patients in remission [50.Van Der Post S. et al.Structural weakening of the colonic mucus barrier is an early event in ulcerative colitis pathogenesis.Gut. 2019; 68: 2142-2151Crossref PubMed Scopus (79) Google Scholar]. Moreover, further studies have revealed that active UC patients and those in remission exhibit decreased intercrypt GCs, leaving areas of the epithelium uncovered and exposed [44.Nyström E.E.L. et al.An intercrypt subpopulation of goblet cells is essential for colonic mucus barrier function.Science. 2021; 372eabb1590Crossref PubMed Scopus (10) Google Scholar]. Studies in animal models have shown that a high demand for MUC2 synthesis leads to endoplasmic reticulum (ER) stress and GC apoptosis, which in turn leads to reduced mucin production and release [51.Heazlewood C.K. et al.Aberrant mucin assembly in mice causes endoplasmic reticulum stress and spontaneous inflammation resembling ulcerative colitis.PLoS Med. 2008; 5e54Crossref PubMed Scopus (493) Google Scholar,52.Tawiah A. et al.High MUC2 mucin expression and misfolding induce cellular stress, reactive oxygen production, and apoptosis in goblet cells.Am. J. Pathol. 2018; 188: 1354-1373Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar]. Although high demand for MUC2 production is thought to follow mucus discharge induced by microbial stimulation or inflammation, the specific factors initiating this process remain unclear. Indeed, in contrast to type 2 T helper cell (Th2) cytokines, regulation of mucus production by Th1 and Th17 cytokines is more controversial, and further work will be necessary to understand the underlying mechanisms and level of regulation. Although colitis (including IBD) and food allergies are etiologically distinct diseases, they often coexist and may share similar pathological mechanisms [53.Virta L.J. et al.Cow's milk allergy, asthma, and pediatric IBD.J. Pediatr. Gastroenterol. 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Although TNF-α is considered to affect epithelial permeability principally by downregulating the expression of tight junction proteins, its impact on intestinal GCs and mucus secretion has not been studied extensively. In mouse pups, TNF-α induced mucus secretion in a TNFRI-dependent manner and Muc2 mRNA expression in a TNFRII-dependent manner [58.McElroy S.J. et al.Tumor necrosis factor receptor 1-dependent depletion of mucus in immature small intestine: a potential role in neonatal necrotizing enterocolitis.Am. J. Physiol. Gastrointest. Liver Physiol. 2011; 301: 656-666Crossref PubMed Scopus (0) Google Scholar]; in addition, these two pathways developed differentially during the postnatal period. Cytokine-induced mucus depletion in the immature ileum emerged through TNFRI-mediated secretion, whereas TNFRII-mediated replenishment was effective only in the mature ileum [58.McElroy S.J. et al.Tumor necrosis factor receptor 1-dependent depletion of mucus in immature small intestine: a potential role in neonatal necrotizing enterocolitis.Am. J. Physiol. Gastrointest. Liver Physiol. 2011; 301: 656-666Crossref PubMed Scopus (0) Google Scholar]. This study highlights the level of complexity when studying MUC2 expression, production, and secretion, and emphasizes the need to explore mRNA as well as protein expression levels. In human mucus-secreting colonic cancer cell lines, TNF-α was reported to induce Muc2 mRNA expression [59.Iwashita J. et al.mRNA of MUC2 is stimulated by