TY - JOUR
AU1 - Etienne-Mesmin,, Lucie
AU2 - Chassaing,, Benoit
AU3 - Desvaux,, Mickaël
AU4 - De Paepe,, Kim
AU5 - Gresse,, Raphaële
AU6 - Sauvaitre,, Thomas
AU7 - Forano,, Evelyne
AU8 - de Wiele, Tom, Van
AU9 - Schüller,, Stephanie
AU1 - Juge,, Nathalie
AU1 - Blanquet-Diot,, Stéphanie
AB - ABSTRACT A close symbiotic relationship exists between the intestinal microbiota and its host. A critical component of gut homeostasis is the presence of a mucus layer covering the gastrointestinal tract. Mucus is a viscoelastic gel at the interface between the luminal content and the host tissue that provides a habitat to the gut microbiota and protects the intestinal epithelium. The review starts by setting up the biological context underpinning the need for experimental models to study gut bacteria-mucus interactions in the digestive environment. We provide an overview of the structure and function of intestinal mucus and mucins, their interactions with intestinal bacteria (including commensal, probiotics and pathogenic microorganisms) and their role in modulating health and disease states. We then describe the characteristics and potentials of experimental models currently available to study the mechanisms underpinning the interaction of mucus with gut microbes, including in vitro, ex vivo and in vivo models. We then discuss the limitations and challenges facing this field of research. intestinal mucus, gut microbiota, experimental models, mucin O-glycosylation INTRODUCTION The human gastrointestinal (GI) tract harbours a complex and diverse community of microbes, including 10 trillion of microorganisms, collectively referred to as the gut microbiota (Sender, Fuchs and Milo 2016). Several regulatory mechanisms cooperate to maintain intestinal homeostasis and a disturbance of the relationship between the gut microbiota and the host can result in several disorders including chronic inflammatory diseases and metabolic syndromes (Rooks and Garrett 2016). While the intestinal microbiota provides important benefits to the host, such as calorie extraction and immune system maturation, it also holds the power to activate various innate and adaptive immune signalling which can lead to uncontrolled and deleterious intestinal inflammation (Pickard et al. 2017). A key component in maintaining a beneficial relationship between the commensal microbes inhabiting the intestine and the host is the presence of an appropriate barrier that prevents bacteria to reach and persist on the epithelial surface (Johansson and Hansson 2016; Sicard et al. 2017; Bretin, Gewirtz and Chassaing 2018). It is well acknowledged that intestinal epithelial cells (IECs) provide a physical and biochemical barrier that prevents the translocation of commensal bacteria to the underlying host tissue. In addition, there is an emerging paradigm that the mucus layer is an important modulator of human health in mediating the homeostatic relationship between the gut microbiota and the host. On the luminal side, the mucus layer provides the first physical, chemical and biological line of defence against large particles, including commensal bacteria and invading pathogens, segregating them from IECs (Turner 2009; Peterson and Artis 2014). Furthermore, mucus provides a biological niche for a microbial community, referred to as mucus-associated microbiota, which is likely to have a major influence on human health (Martens, Neumann and Desai 2018). However, advances in this field of research have been hampered by the lack of suitable model systems recapitulating all the interactions occurring at the mucosal interface. This review provides an overview of currently available experimental models to study the interplay between gut bacteria and intestinal mucus at a mechanistic level, and summarizes their main applications and the challenges remaining in this field of research. OVERVIEW OF MUCUS STRUCTURE AND FUNCTION IN THE GASTROINTESTINAL (GI) TRACT Mucus structural organisation Mucus structure Mucus is a highly hydrated gel made up of more than 98% water that makes it totally transparent, microscopically invisible and difficult to study. This aqueous viscoelastic secretion also contains electrolytes, lipids and various proteins (Bansil and Turner 2018). Mucus is found throughout the entire GI tract from the stomach to the large intestine, with its thickness and structure varying depending on the location, reflecting its various protective functions. The mucus in the small intestine consists of one layer, while the stomach and colon have a bi-layered mucus. In human stomach, the mucus is about 200–400 μm in thickness and consists of an inner layer loosely attached to the epithelial surface, keeping the surface neutral (pH 7) while the gastric lumen pH is acidic (pH 2), and an outer layer which is mobile on the luminal side. Only few bacteria have evolved strategies to colonise the stomach, among which Helicobacter pylori is a specialist (Atuma et al. 2001; Juge 2012). In the small intestine, mucus fills up the space between the villi but is not attached to the epithelium and is somewhat permeable to bacteria (Atuma et al. 2001). In the colon, the two layers mediate opposite interactions with the microbiota; whereas the outer layer (up to 800 μm) is densely colonised by an important microbial biomass, the inner layer (> 200 μm in humans) is virtually devoid of bacteria leaving a space virtually free of microbes (commensals and or pathogens) leaving a space virtually free of microbes above the epithelium (Johansson, Sjovall and Hansson 2013). However, single-cell imaging at tissue scale in mice revealed the presence of bacteria in close proximity of the epithelium (Earle et al. 2015). Among commensal microorganisms, Segmented Filamentous Bacteria (SFB) are immunomodulatory commensals with the ability to adhere to IECs and to invade this mucus layer without invading the host (Hedblom et al. 2018; Ladinsky et al. 2019). Of note, a recent study revealed differences in mucus organization between the proximal and distal colon of rodents (Kamphuis et al. 2017): in the later, the mucus layer is attached to the faecal pellet and absent from the surface of the epithelium (Kamphuis et al. 2017). Other studies demonstrated that the mucus thickens as the microbiota become more diverse, as particularly evident in the colon (Jakobsson et al. 2015). This is also supported by studies using germ free mice showing an impairment in mucus structure (Johansson et al. 2008; Johansson, Sjovall and Hansson 2013; Jakobsson et al. 2015). Gnotobiotic mice colonized with human faecal microbiota present a mucus layer structure resembling that of conventional mice by day 7 post-colonization (Hayes et al. 2018). Animals housed in distinct rooms of the same animal facility exhibit distinct microbiota profiles that are associated with large differences in the inner colon mucus layer, thereby affecting mucus barrier properties (Jakobsson et al. 2015). Also, it has been demonstrated in mice that mucus becomes thinner with age (Elderman et al. 2017). Variations in the mucus thickness and spatial organisation of the gut microbiota in mice were also found to be dependent of the diet (Earle et al. 2015). Interestingly, the thickness of the mucus layer has been shown to undergo circadian fluctuations, with highest microbial proximity to the mucosal surface during the dark phase (Thaiss et al. 2016). Mucus secretion The mucus is produced and secreted by specialized cells namely goblet cells located in the crypt in the small intestine and in higher numbers in the upper crypt in the colon (Johansson and Hansson 2013; Johansson and Hansson 2016; Sicard et al. 2017). Before secretion in the gut lumen, mucin polymers are stored in mucin granulae within the goblet cells (Johansson, Larsson and Hansson 2011; Johansson, Sjovall and Hansson 2013). The function of goblet cells varies depending on their localisation in the small intestinal or colonic crypts (Pelaseyed et al. 2014). Apart from their role in secreting mucus, small intestinal goblet cells can play a role in delivering luminal material to the immune system (Pelaseyed et al. 2014). Interestingly, a study from Gunnar Hanson's laboratory identified a subpopulation of goblet cells called ‘sentinels’ goblet cells (Birchenough et al. 2016). These cells are able to sense Toll-like receptor (TLR) microbial ligands at the entrance of colonic crypts and trigger the activation of NLRP6 inflammasome, leading to mucus secretion from neighbouring goblet cells to defend the colon against bacterial invasion (Birchenough et al. 2016). Renewal of the mucus is an important factor to preserve epithelial damage and bacterial exposure. The colonic mucus has a rapid turnover, since the inner mucus layer is renewed within 1 hour (Johansson 2012), while the gut epithelium renewal takes around 4–5 days (De Weirdt and Van de Wiele 2015). Gastro-intestinal mucins The main structural components of mucus are large glycoproteins called mucins. The protein sequences of mucin domains share a common core structure rich in the amino acids prolin (P), threonine (T) and serine (S) called the PTS domain. These domains are then decorated by O-linked glycans made up of N- acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc), galactose (Gal) and usually terminated by sialic acid and fucose (Juge 2012; Johansson and Hansson 2016; Sicard et al. 2017). These O-glycans render the mucin domains highly resistant to protease degradation and confer mucins their high-water binding capacity. Mucins are produced as transmembrane mucins or secreted gel-forming mucins (Juge 2012; Johansson, Sjovall and Hansson 2013). In the stomach, MUC1 and MUC5AC are produced by the superficial epithelium, while MUC6 are secreted by the stomach glands (Johansson, Sjovall and Hansson 2013; Johansson and Hansson 2016). In the small intestine and colon, mucus is structurally built around the mucin-2 glycoprotein (MUC2). The folding and dimerization of MUC2 is a demanding process owing to the large number of disulfide bonds, and a defect during this process may affect the structure and function of intestinal mucus (Johansson, Larsson and Hansson 2011). Proteolytic cleavages of MUC2 catalysed by the host as well as bacteria enzymes favour the transition from firm to loose layer form and allow bacteria to penetrate into the mucin net-like structure of the outer mucus layer (Johansson et al. 2008). In addition to this proteolytic activity, the degradation of mucin glycan chains by bacterial glycosidases contribute to the establishment of a microbial community in the outer mucus layer (Johansson et al. 2008; Pelaseyed et al. 2014). Mucin glycosylation Glycosylation is the most frequent post-translational modification of proteins and can occur in N-linked and O-linked form, and O-glycosylation is the main modification of mucins (Arike and Hansson 2016). Mucin-type O-glycans are built from eight core structures, with core 1, core 2, core 3 and core 4 glycans most commonly found in intestinal mucins (Brockhausen, Schachter and Stanley 2009). O-glycosylation is initiated in the Golgi apparatus by the addition of a GalNAc residue to the hydroxyl group of serine and threonine of the mucin backbone. Further elongation and branching of the O-glycan chains is governed by a large family of glycosyltransferase enzymes (Bennett et al. 2012). The oligosaccharides can be further modified by addition of histo-blood group antigens (ABO, Lewis), secretor (H) epitopes and sialic acids and sulfate (Rossez et al. 2012; Bansil and Turner 2018). Mucin glycosylation varies along the GI tract (Robbe et al. 2003; Robbe et al. 2004; Holmen Larsson et al. 2013) and is linked to microbial colonization (Juge 2012; Bergstrom and Xia 2013; Tailford et al. 2015; Arike, Holmen-Larsson and Hansson 2017). Mounting evidence suggests that mucin glycosylation is critical to the biological and physical role played by mucus in the gut by influencing the physico-chemical properties and penetrability of mucus and by modulating the composition of the associated mucus-associated microbiota (see section 1.3). Not surprisingly, an alteration of mucin O-glycosylation profile has been reported in intestinal diseases associated with an impaired gut barrier function such as inflammatory bowel disease (IBD) and colorectal cancer (Larsson et al. 2011; Theodoratou et al. 2014) as also supported by work in animal models (Bergstrom et al. 2017) (see also section 4.5). Mucus function in the gut For decades, mucus has been considered to act as a simple physical barrier protecting the host, but mounting evidence suggests that mucus plays additional biological and immunological roles in maintaining gut homeostasis. The coating gel of mucus is acting, in concert with the immune system, the intestinal epithelium and the gut microbiota, to provide a physical, biological and chemical line of defence against potentially harmful invaders while harbouring a distinct microbial community having a major influence on host health. Throughout the gut, the viscous mucus secretion acts as a lubricant that helps the progress of digestive matter along the GI tract and protects the underlying epithelium from excessive mechanical or chemical stresses. In the stomach, the mucus coating creates a pH gradient that protects the epithelium against the crude acidic gastric environment. Mucus acts as a size exclusion filter for larger compounds while selectively allowing transport of small molecules such as gases, ions, nutrients and many proteins to reach the enterocytes (De Weirdt and Van de Wiele 2015), but the mucus lining would prevent digestive enzymes from attacking these cells. In the colon, the outer mucus layer serves as a biological habitat for various microorganisms. Indeed, the glycan structures in the mucus provide potential binding sites and constitute a carbon and energy source to support the growth of commensal but also pathogenic bacteria (Tailford et al. 2015) (see sections 2.1 and 2.2). It is believed that the mucin glycosylation patterns along the GI tract contribute to the microbial tropism of certain taxa in the mucus (Tailford et al. 2015). The mucus layer also helps in the protection of the epithelium and, in association with the immune system, plays a crucial role in intestinal homeostasis. This gel is an important retention matrix for non-mucin proteins with immune regulatory molecules such as antimicrobial molecules (e.g. bactericidal RegIIIγ, α-defensins, secretory immunoglobulins IgAs, etc), therefore limiting the number of bacteria that can reach the epithelium and the underlying immune system (Peterson and Artis 2014; Johansson and Hansson 2016). This physical and biological barrier helps to keep the tremendous amount of bacteria that reside in the lumen as well as enteric pathogens at a safe distance from the epithelium (Chassaing, Ley and Gewirtz 2014; Johansson et al. 2014; Chassaing et al. 2015a). However, this system can be subverted and invading pathogens or pathobionts have evolved strategies to circumvent this barrier by e.g. degrading mucins and/or influencing mucin secretion (Rolhion and Chassaing 2016). In summary, mucus has a dual role in relation to the gut microbiota, it is an ecological niche for bacteria by providing adhesion sites and nutrients, while protecting the underlying epithelium from microbial aggressors that can breach this barrier. The mucus-associated microbiota The gut microbiota composition is known to differ along the longitudinal axis of the GI tract but it also varies transversally from the lumen to the mucosa due differences in key physiological parameters such as nutrient availability or oxygen gradient. The colonic epithelium is made of crypts with specific oxygen conditions and various concentrations of glycans that is a niche for mucin‐degrading bacteria such as Bacteroides fragilis (Pereira and Berry 2017). The use of Carnoy fixative to preserve the mucus layer has been a crucial step for the detection of bacteria in the mucosal environment (Johansson et al. 2008). It is now well appreciated that the faecal microbiota community differs from the luminal, mucosa- or mucus-associated bacterial communities (Swidsinski et al. 2005; Li et al. 2015). Studies in humans demonstrated that the abundance of Bacteroidetes appears to be higher in faecal/luminal samples than in the mucosa (Eckburg et al. 2005). Members of Firmicutes phylum and in particular Clostridium cluster XIVa are significantly enriched in the mucus layer compared to the lumen (Van den Abbeele et al. 2013). Analysis of human colonic biopsies have also shown a distinct mucosal community enriched in Actinobacteria and Proteobacteria compared to the luminal community (Albenberg et al. 2014). Certain species such as Bacteroidesacidifaciens, B. fragilis and Akkermansia muciniphila are enriched in the outer layer of colon mucus (Derrien et al. 2004; Donaldson, Lee and Mazmanian 2016). Similar findings have been observed in animals. Indeed, mice studies have shown that Firmicutes were enriched in the mucosa-associated microbiota, especially members of the Lachnospiraceae and Ruminococcaceae families (Tailford et al. 2015). Bacterial species such as Bacteroides thetaiotaomicron or Escherichia coli display specific genomic repertoires to persist in the outer mucus layer compared with the same species in the intestinal lumen (Li et al. 2015). This spatial localisation may be reflective of the radial oxygen gradient that shapes the mucus-associated and faecal microbiota, since oxygen can favour or impede certain microorganisms (Albenberg et al. 2014). Moreover, laser capture microdissection (LCM) in combination with metagenomics studies provided new insights into the composition of the mucus-associated microbiota (Wang et al. 2010). The use of LCM in mouse models revealed that this microbial community is especially dominated by Acinetobacter in the colonic crypts (Pedron et al. 2012). Using LCM coupled to DNA sequencing based analysis, Chassaing and Gewirtz recently reported profound differences at the phyla level between the inner mucus communities comprising 20% – 60% Proteobacteria and a concomitantly marked reduction in Bacteroidetes as compared to faecal microbiota (Chassaing and Gewirtz 2019). Due to a high polysaccharide content (up to 80% of the mucin biomass), mucus provides an ecological niche for the intestinal microbiota. Mucus-associated bacteria are able to use oligosaccharides from mucins as binding sites through specific bacterial adhesins that promote their colonisation (Section 2.1) or as an energy source to support their growth (Section 2.2). Robbe and colleagues first suggested that the important repertoire of potential ligands and/or carbon sources in mucins could explain the pattern of bacterial colonisation in the different gut regions (Robbe et al. 2004). Mucin degradation has been extensively studied in pathogenic bacteria and more recently investigated in commensal bacteria including A. muciniphila, Bacteroides spp., Bifidobacteria and Ruminococcus spp. (Derrien et al. 2004; De Weirdt and Van de Wiele 2015). A disproportion of bacterial taxa able to invade mucus could further play a role in the development of the dysbiotic microbiota associated with the onset of various intestinal diseases (see section 3). MUCIN-BACTERIA INTERACTIONS Mechanisms of mucin binding by commensal and pathogenic microorganisms in the gut Cell-surface proteins of pathogens and probiotics/commensal strains have been implicated in mediating the binding of microbes to intestinal mucus (Fig. 1). These include (i) specialized cell-surface adhesins or lectins, (ii) appendages such as pili and flagella or (iii) moonlighting proteins (see (Juge 2012) for a review). In particular, a considerable amount of research has been devoted to the characterization of these adhesins in Lactobacillus species (as extensively reviewed in (Van Tassell and Miller 2011; Nishiyama, Sugiyama and Mukai 2016)). Figure 1. Open in new tabDownload slide Mucin-bacterial interactions in the digestive tract.Left panel: Mucins display various and diverse oligosaccharide structures representing potential binding sites for microbial adhesion. Commensal and pathogenic microbes can use cell-surface appendages, such as pili, flagella or fimbriae or adhesins to bind to mucus.Right panel: Mucin glycans are an important energy source for microbes inhabiting the mucus niche that further confer them with an ecological advantage over other members of the gut microbiota. Commensal and pathogenic microorganisms can degrade mucin glycan chains leading to the release of mono- or oligosaccharides from that can be subsequently metabolized by other gut microbes in the mucosal environment. Figure 1. Open in new tabDownload slide Mucin-bacterial interactions in the digestive tract.Left panel: Mucins display various and diverse oligosaccharide structures representing potential binding sites for microbial adhesion. Commensal and pathogenic microbes can use cell-surface appendages, such as pili, flagella or fimbriae or adhesins to bind to mucus.Right panel: Mucin glycans are an important energy source for microbes inhabiting the mucus niche that further confer them with an ecological advantage over other members of the gut microbiota. Commensal and pathogenic microorganisms can degrade mucin glycan chains leading to the release of mono- or oligosaccharides from that can be subsequently metabolized by other gut microbes in the mucosal environment. Mucus binding proteins Mucus-binding proteins (MUBs) containing a variable number of Mub repeats are unique to gut inhabiting Lactobacilli and these proteins have been thoroughly characterised in Lactobacillus reuteri, a gram-positive bacterial species inhabiting the GI tract widely used as a probiotic (Frese et al. 2011). MUB from L. reuteri ATCC 53608 is one of the best-studied examples of mucus adhesins in commensal bacteria. It is a large protein consisting of six type 1 repeats (Mub1) and eight type 2 repeats (Mub2) with each repeat divided into a mucin binding (MucBP) domain and an immunoglobulin binding protein domain (Kuznetsova ; MacKenzie et al. 2009; Etzold et al. 2014b). The Mub repeats mediate binding to mucin glycans, through interactions with terminal sialic acid (Etzold et al. 2014a; Gunning et al. 2016), and Igs (MacKenzie et al. 2009). MUB has the shape of a long, fibre-like structure, of around 180 nm in length (Etzold et al. 2014b), and forms appendices reminiscent to pili found in pathogenic and, more rarely, other commensal bacterial species. However, in contrast to pathogenic pili which adhesin is restricted to the N-terminal tip, MUB interactions with mucin glycans occur through its long and linear multi-repeat structure, as shown by atomic force spectroscopy (Gunning et al. 2016). This multivalent binding would restrict penetration through mucus and limit access of the bacteria to the epithelium surface. In addition, MUB from L. reuteri ATCC 53608 was recently shown to modulate inflammatory responses in human monocyte-derived dendritic cells via interaction with DC-SIGN (Bene et al. 2017). The presence of mucus adhesins was also shown to mediate the binding of L. reuteri strains to both HT-29 and mucus-producing LS174T cells. The binding of L. reuteri to mucus led to a decreased enteropathogenic E. coli (EPEC) adherence to small intestinal biopsy epithelium (Walsham et al. 2016). Recombinant Mub proteins containing Mubs5s6 domains from Lp-1643 protein of L. plantarum Lp9 have been shown to adhere to human intestinal tissue sections (Singh et al. 2017) and inhibited the adhesion of enterotoxigenic E. coli (ETEC) to cultured intestinal HT-29 and Caco-2 cell lines, probably through the recognition of cell-surface mucins (Singh et al. 2018). Together, these findings show that the nature and function of these adhesins are strain-specific with the potential to target either the epithelium or the mucus layer and compete with pathogens. Flagella Several microorganisms have evolved strategies, in particular extracellular appendages such as flagella, pili and fimbriae, to attach to and to penetrate the mucus layer (Juge 2012). Pili and flagella are large polymeric proteins that form long surface structures involved in bacterial adhesion. Flagella are composed of several thousand copies of flagellin subunits and have been extensively studied in EPEC and enterohemorrhagic E. coli (EHEC) for their role in virulence and motility, but their role in mucus binding remains unclear. The adhesive properties of bacterial flagella to mucus were previously reported for Clostridium difficile where crude flagella, recombinant flagellar FliC and FliD proteins were shown to bind to murine mucus (Tasteyre et al. 2001). In pathogenic E. coli strains, the H6 and H7 flagella EPEC E2348/69 and EHEC EDL933 and their flagellin monomers were shown to bind to mucins and to bovine mucus (Erdem et al. 2007). Further studies then showed that EPEC and EHEC O157:H7 adherence to HT-29 cells is related to mucin-type core 2 O-glycan, facilitating invasion into host cells (Ye et al. 2015; Ye et al. 2015). However, flagella are involved in the ability of these pathogenic strains to cross the mucus layer, conferring a selective advantage in penetrating the mucus layers and reaching the epithelial surface, as demonstrated with Adherent-Invasive E. coli (AIEC) LF82 (G368). It is therefore tempting to speculate that in EPEC and EHEC, the flagella have a preference for cell-surface mucins rather than secreted mucus, in line with their ability to penetrate the mucus layer and attach onto the cell surface before invasion. In the probiotic E. coli strain Nissle 1917, a direct interaction was observed between isolated flagella from EcN and porcine MUC2 and human mucus but not murine mucus. The mucus component gluconate was identified as one receptor for the binding of EcN flagella (Troge et al. 2012). EcN was therefore proposed to confer the probiotic strain the ability to compete for binding sites on host tissue with bacterial pathogens. Pili Pili have been identified in Lactobacillus rhamnosus GG where they confer binding to mucus (Kankainen et al. 2009; von Ossowski et al. 2011) and are predicted to exist in other Lactobacillus species including L. casei and L. paracasei, based on genomics analyses (Douillard et al. 2013; Aleksandrzak-Piekarczyk et al. 2015; Nissila et al. 2017). In L. rhamnosus GG, these are composed of a three-protein complex SpaCBA, which has been involved in adhesion to mucus, IECs, and immunomodulatory interactions with IEC (Lebeer et al. 2012; von Ossowski et al. 2013; Ganguli et al. 2015; Vargas Garcia et al. 2015; Bene et al. 2017). The mucus-binding pili of L. rhamnosus GG shares immunological and functional similarities with those of the clinical Enterococcus faecium strain E1165. The binding of E. faecium E1165 to mucus could be prevented by the addition of the mucus-binding SpaC protein or antibodies against L. rhamnosus GG (Tytgat et al. 2016). Collectively, these studies show the potential of using mucus adhesins from probiotic strains to prevent the binding of enteric pathogens to the host. Although not a resident member of the gut microbiota, several Lactococcus lactis strains have also been shown to exhibit mucus-binding properties through bacterial surface proteins such as mucin-binding proteins and pili (as recently reviewed in (Mercier-Bonin and Chapot-Chartier 2017)). The mechanisms of adhesion have been extensively studied by atomic force spectroscopy demonstrating a comparable role played by these two surface proteinaceous components in adhesion of L. lactis TIL448 to pig gastric mucin (PGM) neutral oligosaccharides under static conditions, whereas a more important contribution of the MUBs than the pili one was observed under shear flow (Le et al. 2013). Other cell surface proteins Other cell surface proteins implicated in the binding of commensal bacteria to mucin include aggregation-promoting factors (APFs) from L. plantarum NCIMB 8826 (Bolonkin 1990) or L. lactis (Lukic et al. 2012; Lukic et al. 2014), mucus-binding protein A (CmbA) from L. reuteri ATCC PTA 6475 (Etzold et al. 2014a; Jensen et al. 2014), Lam29 from L. mucosae ME-340 (Watanabe et al. 2010), mucus adhesion-promoting protein (MapA) from L. fermentum/reuteri 104R (Rojas, Ascencio and Conway 2002), a mucus-binding factor (MBF) from L. rhamnosus GG (von Ossowski et al. 2011; Nishiyama et al. 2015), a MucBP-containing mannose-specific adhesin protein (Msa) from L. plantarum WCFS-1 (Pretzer et al. 2005), a 32-Mmubp from L. fermentum BCS87 (Macias-Rodriguez et al. 2009), an extracellular transaldolase (Tal) from Bifidobacterium bifidum DSM20456 (Gonzalez-Rodriguez et al. 2012) and a recently-characterised serine rich repeat protein (SRRP) from L. reuteri ATCC 53 608 (Sequeira et al. 2018). It is expected that adhesion of these commensal or probiotic bacteria to mucus may favour their persistence within the gut in order to exert their beneficial effects to the host. Furthermore, it was recently suggested that carbohydrate binding modules (CBMs) appended to glycoside hydrolases could contribute to the tropism of gut bacteria to glycan-rich area of mucins in the colon, as shown for Ruminococcus gnavus sialic-acid-specific CBM40 (Owen et al. 2017). Blood group binding adhesins In addition, several human enteric pathogens bind to human histo-blood group antigens (HBGAs) expressed on the gut mucosa, including Campylobacter jejuni, Norwalk virus and H. pylori. The role of HBGA recognition to mucin binding has been extensively studied in the gastric pathogen H. pylori where Helicobacter adhesins have been reported to play a critical role in the attachment of the pathogen to both the glycosylated gastric epithelial cell surface and to glycosylated mucins. The binding of H. pylori to gastric mucins through blood group binding adhesin (BabA) and sialic acid-binding adhesin (SabA) revealed a complex charge/low pH-dependent mechanism involving four modes of H. pylori adhesion to MUC5B, MUC7 and MUC5AC mucins (Linden et al. 2008; Skoog et al. 2017). More recently, a novel outer membrane protein adhesin named LabA has been identified in H. pylori and shown to bind to LacdiNAc, a structure, which is also expressed on MUC5AC (Rossez et al. 2014). Binding of H. pylori to gastric mucins therefore is determined both by the mucin glycosylation and also by the adhesins expressed by individual strains. A chitin-binding protein GbpA from Vibrio cholerae shown to bind to N-acetyl-D-glucosamine residues of intestinal mucin has been proposed as an important factor mediating intestinal colonisation and pathogenesis by V. cholerae (Bhowmick et al. 2008; Wong et al. 2012). Moonlighting proteins Unexpectedly, several primarily cytoplasmic proteins have been reported to play a role in mucin binding. Due to their dual function, these proteins are referred to as moonlighting proteins (Henderson and Martin 2011; Henderson and Martin 2013; Henderson 2014). In L. acidophilus, L. plantarum and Mycoplasma genitalium for instance, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was clearly demonstrated to play a role in bacterial adhesion and bind mucins (Alvarez, Blaylock and Baseman 2003; Kinoshita et al. 2008; Patel et al. 2016). While the exact domain responsible for mucin binding remains to be elucidated, GAPDH is suggested to play a similar role in other commensal or pathogenic microorganisms (Kinoshita et al. 2013). In L. reuteri, elongation factor-Tu (EF-Tu) was found to bind the PGM when exposed at the bacterial cell surface (Nishiyama et al. 2013). Here, the sulfated carbohydrate moieties of mucins were demonstrated to play a significant role in EF-Tu-mediated bacterial adhesion to PGM and mucosal surfaces (Nishiyama et al. 2013). Proteosurfaceome analyses in a range of microorganisms have revealed a large repertoire of cytoplasmic proteins present at the bacterial cell surface but their implications in binding to various extracellular matrix (ECM) proteins, including mucins, remain to be more systematically investigated (Chagnot et al. 2012; Desvaux, Candela and Serror 2018). Mechanisms of mucin degradation by commensal and pathogenic microorganisms in the gut Several enzymatic activities are required for the degradation of mucins by pathogens or commensal bacteria including glycoside hydrolases (GHs), sulfatases, or proteases (Fig. 1) as described below. Glycoside hydrolases Mucin glycan degradation in bacteria relies on the expression of GHs such as sialidases (GH33), α-fucosidases (GH29, GH95), exo- and endo-β-N-acetylglucosaminidases (GH84 and GH85), β-galactosidases (GH2, GH20, GH42), α-N-acetylglucosaminidases (GH89), endo-β1,4-galactosidases (GH98) and α-N-acetylgalactosaminidases (GH101, GH129) (www.cazy.org). These enzymes have been functionally characterised in resident members of the gut microbiota able to forage on mucins, including A. muciniphila, B. thetaiotaomicron, B. bifidum, B. fragilis and R. gnavus, as recently reviewed (Tailford et al. 2015; Ndeh and Gilbert 2018). The released mono- or oligosaccharides derived from mucus degradation by these commensal bacteria can be utilised by the bacteria itself or scavenged by other bacteria inhabiting the mucus niche including pathogenic species such as Salmonella species, C. difficile, diarrhoeagenic E. coli or Vibrio cholerae through cross-feeding interactions (Fabich et al. 2008; Abyzov et al. 2012; Ng et al. 2013). In addition, some of these pathogens have the glycolytic potential to release mucus-derived sugars for their own consumption (Mondal et al. 2014; Arabyan et al. 2016). Sulfatases Sulfatases are being increasingly investigated for their role in modulating the gut microbial ecosystem in health and disease. Some members of the gut microbiota such as B. thetaiotaomicron, Bacteroides ovatus and Prevotella sp. strain RS2 Bifidobacterium breve UCC2003, or B. fragilis possess mucin‐desulfating sulfatases or glycosulfatases (Salyers et al. 1977; Berteau et al. 2006; Benjdia et al. 2011; Egan et al. 2016; Praharaj et al. 2018). Mucin sulfatase activity of these species may provide them a competitive advantage in the infant gut and/or the adult gut. The mucin‐desulfating sulfatases that have been characterised so far include sulfatases specific for the ‐D‐galactopyranosyl 3‐sulfate, ‐Dgalactopyranosyl6‐sulfate and 2‐acetamido‐2‐deoxy‐D‐glucopyranosyl6‐sulfate (6‐SO3‐GlcNAc) building blocks of the oligosaccharide chains. GlcNAc-6-S can be found in terminal or branched positions of mucin oligosaccharide. The desulfation of mucin by bacterial sulfatases may be a rate-limiting step in mucin-degradation mechanism, allowing glycosidases to access and act on the mucins by other members of the gut microbiota. The release of sulfate from mucins may also contribute to the expansion of Sulfate-reducing bacteria (SRB) in the gut (Rey et al. 2013). SRB are able to produce hydrogen sulfide (H2S) which can reduce disulfide bonds present in the mucus network, leading to mucus erosion and access of bacteria to the epithelium, therefore contributing to epithelial damage and inflammation This mechanism has been proposed to be involved in the aetiology and/or severity of IBD (Ijssennagger, van der Meer and van Mil 2016). In addition, Hickey and colleagues showed that sulfatases of B. thetaiotaomicron are required for its outer membrane vesicles to transit to underlying host immune cells and cause colitis (Chatzidaki-Livanis and Comstock 2015). Together these data highlight the complex role of bacterial sulfatases in the gut. Proteases Bacterial proteases from commensal or pathogenic E. coli have also been implicated in the recognition and degradation of mucins. In EHEC, StcE (secreted protease of C1 esterase inhibitor from EHEC) was originally described as specifically cleaving C1 esterase inhibitor (C1-INH) (Lathem et al. 2002; Grys, Walters and Welch 2006) but later showed to be even more active against MUC7 (Lathem et al. 2002). This soluble enzyme is important in reducing mucin levels. StcE has been suggested to have a dual role during human infection, (i) by promoting the penetration of bacterial cells through the mucus barrier lining the GI tract and thus facilitating the intimate EHEC adherence to IECs, which is an essential step in colonisation (Hews et al. 2017), and (ii) by acting as an anti-inflammatory agent protecting bacterial and host cell surfaces from complement-mediated lysis (Grys et al. 2005; Abreu and Barbosa 2017). StcE is secreted by a Type II, subtype a, secretion system (T2aSS) (Monteiro et al. 2016; Hay et al. 2018). This mucinase is a metalloprotease belonging to the peptidase M66 family (IPR019503) carrying one zinc atom per protein but no structural calcium, which is a reported feature of metalloproteases (Yu, Worrall and Strynadka 2012). Recently, EHEC StcE metalloprotease was shown to reduce the inner mucus layer in human colonic mucosal biopsies and the MUC2 glycoprotein levels in mucin-producing LS174T colon carcinoma cells (Hews et al. 2017). Pic (protein involved in intestinal colonisation), also previously known as Shmu (Shigella mucinase), is a secreted protease identified in Shigella flexneri and enteroaggregative E. coli (EAEC) (Henderson et al. 1999a). Pic is secreted by a Type V, subtype a, secretion system (T5aSS) and belongs to the subfamily of serine protease autotransporters (SPATEs), with a catalytic domain corresponding to the peptidase S6 family (IPR030396). This enzyme was reported to display proteolytic activity against gelatin as well as bovine and murine mucin but not hog gastric mucin (Henderson et al. 1999a). PicU was also shown to exhibit mucinolytic activity in uropathogenic E. coli (Parham et al. 2004). Hbp (hemoglobin-binding protease), also previously known as Tsh (temperature-sensitive haemagglutinin), is capable of cleaving bovine submaxillary mucin but not hog gastric mucin, which so far would appear as a feature of mucinolytic serine protease autotransporter of Enterobacteriaceae (SPATE) of the peptidase S6 family (Dutta et al. 2002). In some EHEC strains, a SPATE of the peptidase S6 family exhibiting mucinolytic activity was identified on plasmid pO113, namely EpeA (EHEC plasmid-encoded autotransporter) (Leyton et al. 2003). In AIEC, a Vat (vacuolating autotransporter) homologue belonging to the SPATE of the peptidase S6 family was demonstrated to exhibit a mucinolytic activity (Gibold et al. 2016). Vat-AIEC appears to significantly contribute to the colonisation ability of AIEC by decreasing mucus viscosity as well as enhancing bacterial penetration in mucus and access to IECs (Gibold et al. 2016). In some non-O157 EHEC strains, a subtilase cytotoxin (SubAB) was identified (Paton et al. 2006; Wang, Paton and Paton 2007) and appeared to contribute to mucin depletion as shown with a Shiga-toxin encoding E. coli (STEC) O113:H21 strain (Gerhardt et al. 2013). While the A subunit harbours the enzymatic activity with a subtilase-like serine protease domain belonging to the peptidase S8/S53 family (IPR000209), the mucinolytic activity of SubAB remains to be clearly established. Other proteins have been described in V. cholerae. Among them, TagA is a secreted protease of V. cholerae that specifically cleaves mucin glycoproteins (Szabady et al. 2011). The V. cholerae extracellular chitinase ChiA2 secreted in the intestine hydrolyzes intestinal mucin to release GlcNAc, and the released sugar is successfully utilized by V. cholerae for growth and survival in the host intestine (Mondal et al. 2014). SslE (secreted and surface associated lipoprotein), previously known as YghJ, is a secreted and cell-surface lipoprotein degrading the major mucins in the small intestine, namely MUC2 and MUC3, thus facilitating bacterial penetration of the mucus layer and ultimately adhesion to host cells (Luo et al. 2014; Valeri et al. 2015; Tapader, Bose and Pal 2017). SslE is secreted via a T2aSS and appears inactive against the mucin-like CD43, bovine submaxillary mucin, gelatin, or IgG (Luo et al. 2014). This Zn-metalloprotease, belonging to the peptidase M60 family (IPR031161), is found in pathogenic and commensal E. coli, including ETEC, EHEC O104:H4, E. coli SE-11 or Nissle 1917 strains. AcfD (accessory colonisation factor D) from V. cholerae is homologous to SslE but its putative mucinolytic activity remains to be investigated (Peterson and Mekalanos 1988). Of note, SslE is also considered as a relevant target for the development of vaccines against intestinal pathogenic E. coli (Nesta et al. 2014; Naili et al. 2016; Naili et al. 2017). Importance of mucus-bacteria interactions in health and disease In the colon, the outer mucus layer offers a niche to commensal bacteria by providing preferential binding sites (Section 2.1) and nutrients (Section 2.2). Due to its proximity to host cells and the immune system, the mucus-associated microbiota, sometimes also referred to as the mucobiome (Belzer et al. 2017), has been proposed as an important modulator of health. The integrity of the mucosa relies on a combination of factors including the gut microbiota composition, the diet and host genetic factors (Fig. 2) (Martens, Neumann and Desai 2018). The mucus and mucus-associated bacterial community play a key role in limiting access of invading pathogens to the underlying epithelial cells and in limiting the progression of intestinal and extra-intestinal diseases (Donaldson, Lee and Mazmanian 2016). Figure 2. Open in new tabDownload slide Perturbations of the mucus barrier in response to environmental and microbial stimuli.This figure represents an overview of the various factors (diets, nanomaterials, pollutants, antibiotics or invading pathogens) affecting the gut microbiota composition and/or the thickness, structure and composition of the mucus barrier. Disruption of the mucus layer promotes bacterial encroachment leading to the subsequent development of low-grade inflammation, associated with inflammatory bowel diseases and metabolic disorders. Figure 2. Open in new tabDownload slide Perturbations of the mucus barrier in response to environmental and microbial stimuli.This figure represents an overview of the various factors (diets, nanomaterials, pollutants, antibiotics or invading pathogens) affecting the gut microbiota composition and/or the thickness, structure and composition of the mucus barrier. Disruption of the mucus layer promotes bacterial encroachment leading to the subsequent development of low-grade inflammation, associated with inflammatory bowel diseases and metabolic disorders. Effect of bacteria and bacterial products on mucus production A number of animal studies (using antibiotic-treated, germ-free or gnotobiotic mice) suggest that the presence of bacteria triggers the development of the protective mucus layer. Mice treated with the antibiotic metronidazole, but not streptomycin, display an altered goblet cell function and thinning of the inner mucus layer (Wlodarska et al. 2011). However, another study reported that depletion of the intestinal microbiota following a 3 week-antibiotic period (cocktail of four antibiotics) did not modify mucus penetrability (Johansson et al. 2015). Compared to conventionally housed animals, germ-free mice have fewer goblet cells, which are smaller in size (Kandori et al. 1996) and harbour an impaired mucus layer, indicating that the formation of the protective mucus layer depends upon the presence of bacteria (Rodriguez-Pineiro and Johansson 2015). Johansson and colleagues demonstrated that the mucus of germ-free mice displayed a significant decrease in Muc2 level and was more penetrable to bacterium-size fluorescent beads as compared to conventionally raised mice (Johansson et al. 2015). The gut microbiota composition of germ free animals is normalized two weeks after colonisation in terms of microbiota composition, but up to 8 weeks are needed to reach a normalized mucus phenotype (Johansson et al. 2015; Hayes et al. 2018). In support of this, fortification of the mucus layer and increased diversity of mucin glycosylation was observed within 48  hours of human intestinal organoid colonization with human-derived, non-pathogenic E. coli (Hill et al. 2017). Some bacteria, in particular Anaerostipes, have been shown to display mucus-stimulating properties (Jakobsson et al. 2015). Lactobacillus species can also stimulate MUC2 production and secretion by the goblet cells in the human gut (Sicard et al. 2017). Representative members of the two main phyla of the gut microbiota, B. thetaiotaomicron and Faecalibacterium prauznitzii can modulate goblet cell differentiation and thus mucus production (Wrzosek et al. 2013). A recent study showed that Streptococcus thermophilus, a transient food-borne bacterium, was able to induce mucus pathway in gnotobiotic rodents despite its poor capacity for mucus adhesion and mucin glycan degradation in vitro (Fernandez et al. 2018). Some of the mechanisms mediating mucin production and secretion by gut bacteria have been elucidated as described below. Pathogen associated molecular patterns such as lipopolysaccharide (LPS) or peptidoglycan are known to induce mucus production (Petersson et al. 2011). LPS and flagellin purified from Gram-negative bacteria as well as lipoteichoic acid from Gram-positive bacteria have been shown to induce mucin upregulation via the Ras-signalling pathway (McNamara and Basbaum 2001). LPS also increases the production of interleukin (IL)-8 by goblet cells, which further promotes mucin secretion (Smirnova et al. 2003). TLR family members play an important role in mucus formation. Mice lacking the TLR adaptor protein MyD88 show a decreased production of mucus (Bhinder et al. 2014). Mice engineered to lack the flagellin receptor, TLR5 deficient mice, have a disorganised mucus layer and lack a well-defined inner layer when compared to wild type animals with an increase abundance of Proteobacteria in close contact with the epithelial surfaces (Carvalho et al. 2012; Chassaing, Ley and Gewirtz 2014; Chassaing et al. 2015a). Lastly, it has been shownin vitro using various human-derived cell lines that bacterial metabolites such as short-chain fatty acids (SCFA) and especially butyrate can stimulate MUC2 production in the absence of other energy sources (Willemsen et al. 2003; Gaudier et al. 2004). The effect of butyrate on MUC2 gene expression is mediated by epigenetic modifications (acetylation/methylation of histones) on the MUC2 promoter as demonstrated in vitro using human goblet cell-like LS174T cells (Burger-van Paassen et al. 2009). Fernandez and colleagues suggested that lactate produced by S. thermophilus in the GI tract could stimulate mucus production via a signalling pathway dependent of KLF4, a transcription factor involved in the differentiation of goblet cells (Fernandez et al. 2018). Some other bacterial effectors have been identified to mediate mucin expression and glycosylation such as small peptides from R. gnavus and B. thetaiotaomicron (see section 2). Interactions of pathogens with mucus The mucus barrier provides a bulwark against intestinal pathogens (Johansson, Sjovall and Hansson 2013; Sicard et al. 2017; Martens, Neumann and Desai 2018). The importance of intestinal mucus in controlling enteric infection has been widely documented in Muc2 knockout mice (Muc2−/− mice) (see section 4.5), which do not produce mucus in the small and large intestine, thus leading to a close contact between bacteria and the epithelium. Bergstrom and colleagues reported that Muc2−/− mice exhibit an increase susceptibility to murine bacterial pathogen Citrobacter rodentium (Bergstrom et al. 2010). Likewise, Muc2 plays a crucial role in controlling Salmonella infection (Zarepour et al. 2013). In a similar way, H. pylori has evolved mechanisms allowing its residence in the gastric mucus layer (Moore, Boren and Solnick 2011). As previously described for bacteria, Muc2−/− mice are also more susceptible to enteric parasitic infection with Trichuris muris since they exhibit a delayed expulsion of the parasite compared to wild type animals (Hasnain et al. 2010). Clearance of parasitic infection is associated with exclusion of helminths via a TH2 cell-mediated goblet cell increase and mucus release (Artis and Grencis 2008). Entamoeaba histolytica also possesses lectins binding to mucins and secretes proteases responsible for the cleavage of Muc2, allowing the protozoan to invade the underlying epithelium (Lidell et al. 2006). Recently, a detailed investigation of the cooperative roles for colonic microbiota and Muc2 in mediating innate host defence against E. histolytica was carried out using Muc2−/− mice, germ free mice and mucus-secreting LS174T cells, demonstrating that mucus secretion and pro-inflammatory responses were microbiota-specific (Leon-Coria et al. 2018). Lastly, as shown with S. flexneri and H. pylori, some pathogenic bacteria are able to reshape mucin structures by remodelling their glycosylation pattern in a type III secretion system-dependent manner (Sperandio et al. 2013; Magalhaes et al. 2015). Emerging data suggest that pathogenic bacteria can benefit from the capacity of commensal microorganisms to release mucin degradation products that can be used to support their proliferation within the mucus niche. For example, B. thetaiotaomicron can release free sialic acid from colonic mucus glycans that can be utilized by C. difficile and Salmonella Typhimurium to promote their own colonisation and persistence in the gut (Ng et al. 2013). Another study indicates that EHEC bacteria colonise the mucus layer within the cooperation of local bacterial communities including B. thetaiotaomicron and other anaerobes which are able to cleave host glycan-derived sugar and produce fucose (Pacheco et al. 2012). EHEC then senses fucose produced by B. thetaiotaomicron to control expression of its type III secretion system (Pacheco et al. 2012; Cameron et al. 2018). Mucus-pathogen interactions have also been evidenced in the extra-digestive area. Pseudomonas aeruginosa, a Gram-negative-flagellated pathogen, is the main causal agent for the development of pneumonia in immunocompromised patients and patients with cystic fibrosis (CF). This infection is associated with a genetic disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride and bicarbonate ion channel protein with a key role in protecting the small intestine from bacterial invasion. CF conducts to blockage airway, mucus hypersecretion leading to chronic bacterial lung infections and inflammation. It has been shown that P. aeruginosa, via LPS, upregulate MUC2 and MUC5AC gene expression contributing to the excessive mucus production and airway blockage seen in CF (Bellu et al. 2013). Effect of diet on mucus Recent evidences have demonstrated that the diet can influence the properties of colonic mucus and thereafter interfere with the gut microbiota. Fibres Living in symbiosis with the host, the gut microbiota depends mostly on non-digestible fibres and polysaccharides as energy source. In the absence of fibres in the diet, the gut microbiota shifts towards the utilisation of host glycans such as those provided by mucins, resulting in a thinner protective colonic mucus (Sonnenburg and Sonnenburg 2014; Earle et al. 2015). Accordingly, Desai and colleagues demonstrated that a low-fibre diet promotes the enrichment of mucin-degrading bacteria and the overexpression of carbohydrate-active enzymes (CAZymes) that degrade the colonic mucus barrier (Desai et al. 2016; Martens, Neumann and Desai 2018). In these mice fed with a deprived-fibre diet, infection with C. rodentium promotes greater epithelial access and lethal colitis (Desai et al. 2016). A lack of fermentable fibres in the diet also leads to a reduction in epithelial cell proliferation resulting in a thin mucosa with encroached bacteria (Chassaing et al. 2015b). Further studies in mice reported that inulin supplementation increases the number of colonic goblet cells, which correlates with a thicker mucus layer and an increase proportion of the Akkermansia genus (Kleessen, Hartmann and Blaut 2003; Everard et al. 2013). Similarly, studies in mice showed that a diet enriched in inulin fibre prevents mucus deterioration (Schroeder et al. 2018). It was recently showed that inulin but not cellulose protects against diet-induced obesity by reducing microbiota encroachment in a cytokine IL-22-dependent manner (Zou et al. 2018), demonstrating the importance of dietary factors, especially soluble fibre, in the homeostasis of host–microbiota relationship. Considering the increased mucus foraging activity occurring when mice are fed with a low-fibre diet, a recent study showed that supplementation with probiotic bifidobacteria (B. longum) or prebiotic fibre (inulin) could reduce such mucus defect. Notably, administration with B. longum was sufficient to restore mucus growth, while administration with inulin could prevent the increase of mucus penetrability in mice fed a western style diet (WSD) (Schroeder et al. 2018). Western diet and food additives Besides fibres, other nutrients within a WSD can modulate intestinal barrier function. A WSD is a rich in saturated fats and simple carbohydrates but depleted in dietary fibres. As a result, a diet-induced obesity in mice leads to colon mucosal barrier dysfunction with a thinner mucus layer (as described above) and treatment with A. muciniphila appears to counteract this effect by improving mucus thickness (Everard et al. 2013). Similarly, mice fed a high-fat and high-sugar diet exhibit an increased abundance of mucin-degrading species leading to a decrease in mucus thickness (Martinez-Medina et al. 2014). The diet of modern societies has dramatically changed as evidenced by a steady increase in the consumption of processed foods concomitantly with an increase in the use of food additives (Chassaing et al. 2015a). Mice treated with dietary emulsifiers (polysorbate 80 or carboxymethylcellulose) show a reduced mucus thickness and increased gut permeability. In these animals, some bacteria appear in close contact with the epithelium. Emulsifier-treated mice have an altered microbial composition associated with increased levels of mucolytic bacteria including R. gnavus and a marked reduction in microbial diversity, with a bloom in Verrucomicrobia phyla, especially A. muciniphila (Chassaing et al. 2015a). This may further contribute to the intestinal passage of bacterial constituents such as LPS and flagellin, which participates in the development of low-grade inflammation and metabolic disorders in wild type mice and of colitis in susceptible host animals (Chassaing, Ley and Gewirtz 2014; Chassaing et al. 2015a; Chassaing et al. 2017b). Mice fed with diets enriched in maltodextrin, a filler and thickener used in food processing, show a reduction of Muc2 expression, making the host more sensitive to low-grade inflammation but with no significant change in mucosa-associated microbiota (Laudisi et al. 2018). Effect of food contaminants on mucus The intestinal mucosa is increasingly appreciated as a key player in the emerging field of gut toxicology of environmental pollutants, as recently reviewed (Gillois et al. 2018). Human contamination mainly occurs via the oral route through consumption of food but also through polluted water and soil exposure. Nanomaterials The use of nanotechnology in many common consumer products, especially in food products, is growing. Scarce studies have evaluated the interactions of food nanoparticles with the microbiota and mucus (Mercier-Bonin et al. 2018). It was shown in vitro that common nanoparticles of Titanium dioxide (TiO2) are trapped into mucus, leading to areas with a high local concentration (Talbot et al. 2018). Silver nanoparticles are widely used in food industry to colour the surface of confectionary and pastries. Rats fed with these particles exhibit higher numbers of goblet cells and a modification of the glycosylation pattern of mucins with a decreased proportion of sulfated mucins and an increased proportion of sialyated mucins (Jeong et al. 2010). Repeated silver nanoparticle-exposure may therefore produce pathological regions in the lamina propria (Jeong et al. 2010). Persistent Organic Pollutants A recent study showed that mice chronically exposed to benzo[a]pyrene (BaP) which is the most toxic member of the polycyclic aromatic hydrocarbons family display significant shifts in the composition and relative abundance of stool and mucosa-associated bacterial communities (decrease of Verrucomicrobiaceae, represented by A. muciniphila) (Ribiere et al. 2016). Furthermore, exposure to perfluorooctane sulfonate (PFOS, environmental contaminant used as a surfactant and repellent) in a mouse model of C. rodentium infection led to a significant reduction in mucin gene expression and a failure to clear the bacterial infection (Suo et al. 2017). Smoke exposure also significantly affects the mucosa-associated bacterial community and alters the expression of mucins in the murine gut (Allais et al. 2016). Mucus and inflammatory-related diseases Inflammatory bowel diseases (IBD) Barrier disturbances including alterations in the thickness or composition of the intestinal mucus layer are recognized to play a crucial role in the onset of GI disorders such as Crohn disease (CD) or ulcerative colitis (UC). The mucus layer in UC patients is thinner and has an altered glycosylation profile making it more penetrable to bacteria (Johansson et al. 2014). To better understand the onset of IBD, several murine models of intestinal inflammation (genetically or chemically induced) have been established. The most common experimental model of colitis relies on the administration of Dextran Sodium Sulfate (DSS) in the drinking water. Mice orally administered with DSS display an inner mucus layer which is more penetrable by bacteria within 12 hours (Johansson et al. 2010). Similarly, IL-10−/− and TLR5−/− mice that develop spontaneous colitis have a thicker mucus layer and more penetrable inner mucus layer when compared to wild type animals (Johansson et al. 2014). Muc2−/− mice develop intestinal inflammation with diarrhoea, rectal bleeding and prolapse (Johansson et al. 2008) and are more susceptible to DSS-induced colitis; these animals exhibit a massive number of bacteria in close contact with host tissues, further promoting inflammation (Van der Sluis et al. 2006). Moreover, abnormal mucin O-glycosylation has been associated with an increased inflammation, highlighting the importance of mucin glycans in the maintenance of gut homeostasis (Johansson et al. 2014) (Bergstrom and Xia 2013; Bergstrom et al. 2016). These changes in mucus composition were also mirrored by changes in the gut microbiota composition at the mucosal surface. IBD patients exhibit a disproportion of mucin-degrading (or mucinolytic) bacteria with an increased abundance of Ruminococcus torques and R. gnavus, but a decreased abundance of A. muciniphila. In addition, the expansion of certain pathobionts and in particular AIEC exhibiting mucinolytic activity has been reported to favour gut colonisation and further induce inflammation in CD (Palmela et al. 2018). Taken together, these data suggest that mucus-bacteria interactions contribute to the intestinal barrier dysfunction in IBD patients and future work is needed to better understand the influence or consequence of these interactions on the disease. Obesity and metabolic-related disorders A correlation between adiposity, dysglycemia and microbiota encroachment has been reported in a number of animal studies. Muc2−/− mice fed a High Fat Diet (HFD) are protected from diet-induced weight gain, fatty liver, and insulin resistance as they displayed less inflammation and increased systemic levels of IL-22 (Hartmann et al. 2016). This study supports a role of Muc2 during obesity and highlights the importance of the crosstalk between microbiota, mucus and immune mediators. In mice fed a HFD, mucus secretion is altered in the ileum but not in the duodenum and jejunum, largely in response to an alteration of PPAR-γ signalling. In these mice, Muc2 accumulates at the apical side of goblet cells, leading to a reduction in the expansion capacity of the mucins, thus strongly altering the phenotype of the mucus layer (Tomas et al. 2016). Studies by Chassaing and colleagues in different mouse models of metabolic syndrome and in humans demonstrated that bacteria have the ability to infiltrate the mucus layer and reach the epithelium (Chassaing, Ley and Gewirtz 2014; Chassaing et al. 2015a; Chassaing et al. 2017a). Further, measurement of bacterial-epithelial distance reveals that microbiota encroachment is a feature of insulin resistance-associated dysglycemia in humans that may promote inflammation (Chassaing et al. 2017a). Several studies demonstrated that A. muciniphila is less abundant in the intestinal microbiota of both genetic and diet-induced obese and diabetic mice, as well as in individuals with obesity, when compared to the faecal microbial population of healthy individuals (Everard et al. 2013; Shin et al. 2014). A. muciniphila treatment has been shown to reverse fat gain, serum LPS levels, gut barrier function and insulin resistance. In addition, oral administration of an outer-membrane protein from A. muciniphila led to reduced fat mass and metabolic syndrome in mice fed an obesity-induced diet (Plovier et al. 2017). Conversely, anti-diabetic treatments such as metformin administration led to an increase in the Akkermansia spp. population (Shin et al. 2014). Human studies have shown that alcohol abuse induced alcoholic liver diseases (ALD) are associated with an increase in intestinal mucus thickness in patients, using wheat germ agglutinin staining on duodenal biopsies (Hartmann et al. 2013). Animal studies demonstrated that when compared to wild type animals, Muc-2−/− mice are protected from alcoholic steatohepatitis in an experimental alcohol-induced liver disease model (Hartmann et al. 2013). In addition, Muc-2−/− mice are protected from Non Alcoholic Fatty Liver Disease (NAFLD) when fed a high-fat diet inducing liver steatosis (Hartmann et al. 2016). Altogether, these data highlight the role of mucus and mucins in the gut-liver axis. Cancer The role of mucins in cancer progression has been extensively reviewed (Hollingsworth and Swanson 2004; Kufe 2009). Muc2−/− mice displayed spontaneous development of adenomas in the small intestine that progressed to invasive adenocarcinoma, as well as rectal tumours (Velcich et al. 2002). In humans, high levels of expression of MUC2 by pancreatic and biliary tumours has been associated with a low degree of invasiveness, malignancy and a better prognosis as compared to tumours not expressing MUC2 (Hollingsworth and Swanson 2004). An abnormal mucin O-glycosylation has been associated with an increased inflammation that could contribute to the development of colitis-associated colon cancer in mice (Bergstrom and Xia 2013; Bergstrom et al. 2016). Together these studies support the role of MUC2 as a tumour suppressor. POTENTIAL OF EXPERIMENTAL MODELS TO STUDY MUCUS/MUCIN INTERACTIONS WITH GUT MICROBES As mounting evidences highlight the importance of mucus in the cross-talk between the gut microbiota and the host, a wide range of experimental models has been developed to study mucus-bacteria interactions (Table 1). These include the use of purified mucins, mucin-secreting cells or tissues, or mucin-containing fermentation models, as described below. Table 1. Experimental models available to study mucus-bacteria interactions.GI : Gastro-Intestinal, HMI : Host–Microbe Interactions, IBD : Inflammatory Bowel Ddiseases, IVOC : In vitro organ culture, M-SHIME : Mucus Simulator of the Human Intestinal Microbial Ecosystem. Types of models Description Applications Advantages Limitations References In vitro mucus/mucin binding assays Microplates—Flow chambers * Immobilization of mucus/mucin on the microtiter plate* Microtiter plate: adhesion in static conditions* Flow chambers: adhesion under dynamic conditions (fluid shear) * Evaluation of bacterial adhesion (commensals and pathogens) to mucins and molecular mechanisms associated * Fast, quantitative and high throughput method to study mucus-microbe interactions independently from other in vivo conditions* Identification of molecular determinants involved in adhesion of microbes* Coupling with biophysical techniques (Surface Plasmon Resonance, Atomic Force Microscopy) * Influence of experimental conditions (antibiotics, mechanical treatments, growth conditions, hydrophobic interactions)* Limited availability of purified mucins (mainly use of pig gastric mucin)* Absence of gut microbiota McNamara et al. 2000; Gusils et al. 2004; Ringot-Destrez et al.2018; Clyne et al.2017; Dunne et al. 2018 In vitro cell models Monoculture models *Gut–derived epithelial cells resembling intestinal tissue consisting mainly of mature goblet cells that secrete an adherent *Adherence of commensal and pathogenic bacteria to host cells * Effect of commensals/pathogens on host cell mucin synthesis and/or composition of the mucus layer *Reproducible and easily handled in laboratories* Identification of molecular determinants involved in adhesion of microbes and host cell mucin synthesis * Good platform for screening and characterizing probiotic activity *Derived from cancer cells, different from healthy tissue* Not representative of various cell types recovered in mucosal epithelial tissues* Not representative of appropriate MUC gene expression* Modulation of mucus production by culture conditions * Absence of gut microbiota* Difficulty to maintain for long-term experiments (> 1 month)* Requirement of high oxygen levels (difficulty to study oxygen-sensitive microbes) Linden et al. 2007; Navabi et al. 2013; Hews et al. 2017 Co-culture models *Mixed culture of enterocytes and mucin secreting cells *Adherence of commensal and pathogenic bacteria to host cells * Effect of commensals/pathogens on host cell mucin synthesis and/or composition of the mucus layer *Better representation of cell-type ratio recovered in mucosal epithelial tissues* Simple model, well described in literature *Absence of M-cells (development of triple co-culture Caco-2/HT29-MTX/Raji B) * Variations in seeding ratios of HT29 MTX/Caco-2 can impede results interpretation* Modulation of mucus production by culture conditions * Absence of gut microbiota* Difficulty to maintain for long-term experiments (> 1 month)* Requirement of high oxygen levels (difficulty to study oxygen-sensitive microbes) Hilgendorf et al. 2000; Lozoya-Agullo et al. 2017 Ex-vivo organ cultures Intestinal organoids *Generation of self-propagating spheres of primary intestinal epithelial cells * Enteroids = derived from adult stem cells isolated from the crypts of human small intestinal * Colonoids = derived from adult stem cells isolated from the crypts of human colonic tissue *Study of advanced aspects of mucus development in a more complex scenario* Study of host–commensals and pathogens interactions *Often collected from mice tissues, possible use of patient-derived tissues* Assay that more accurately mimics in vivo conditions* Amenable to long-term culture *Highly expensive and requires specialized expertise* Requires access to biopsies/tissues * Donor-to-donor variability* Requirement of injection to infect organoids with bacteria* Absence of gut microbiota* No reproduction of peristalsis motions and GI stressful events* Requirement of high oxygen levels (difficulty to study oxygen-sensitive microbes) Jung et al. 2011; Sato et al. 2011 In vitro organ culture (IVOC) *Whole organs maintained in vitro *Study of host–commensals and pathogens interactions *Better maintenance of tissue architecture *Presence of non-transformed cells including all major cell types (enterocytes, goblet cells, Paneth cells and endocrine cells)* Often collected from animal tissues, possible use of patient-derived tissues* Possible use of biopsies from disease patients (e.g. IBD) *Requires access to biopsies/tissues * Expensive and requires expertise *Donor-to-donor variability* Difficulty to maintain for long-term experiments* No reproduction of peristalsis motions and GI stressful events* Requirement of high oxygen levels (difficulty to study oxygen-sensitive microbes) Browning and Trier 1969; Schüller et al. 2007 Gut- on-a chip *Reproduction of the multicellular structures, cell–cell and tissue–tissue interactions, and the native microenvironment* Closely reproduction of the in vivo situation *Study of the complex physiological and pathophysiological responses of tissues at an organ level* Study of host–commensals and pathogens interactions *Presence of non-transformed cells including all major cell types (enterocytes, goblet cells, Paneth cells and endocrine cells)* Reproduction of peristalsis like motions * Possible use of biopsies from disease patients (e.g. IBD) *Expensive and requires dedicated expertise and instrumentation* Stem cell differentiation is difficult to achieve* Flow rate of the medium can influence cell metabolism* Absence of gut microbiota* No input from immune and nervous system* Requirement of high oxygen levels (difficulty to study oxygen-sensitive microbes)* No reproduction of the full complexity of the human gut microbiota Kasendra et al. 2018 In vitro human colonic models involving a mucosal phase M-SHIME *Series of bioreactor modeling the different parts of the human gut* Introduction of mucus-coated carriers (Mucus SHIME) *Study the fine-scale spatial organization of the gut microbial ecosystem* Investigation of the interactions between commensals, pathogens, probiotics and luminal/mucosal gut microbiota *Integration of human GI -related parameters and possibility to modulate them depending on diet, age and diseases (e.g. ulcerative colitis)* Capture dynamics by time-resolved analyses* Capture inter-individual variability of human gut microbiota* Possible long term experiments* Possible coupling with cell culture models and Host-Microbe Interactions (HMI) module *Expensive and requires expertise and specialized instrumentation* Use of pig gastric mucin* No reproduction of immune and nervous system* No reproduction of the full complexity of the human gut microbiota* Donor-to-donor variability Van den Abbeele et al. 2012; Van den Abbeele et al. 2013; De Paepe et al. 2018 In vivo animal models *Whole organism models* Development of genetically modified mice with impaired mucin production (comparison with wild type animals) *Study of the functional roles of mucin and mucus under physiological or pathological conditions at the level of entire organism* Investigation of downstream consequences of mucin modulation in mucosal barrier defense* Investigation of the interactions between commensals, pathogens, probiotics and luminal/mucosal gut microbiota *Physiological model* Allow targeting of a specific gene/pathway in the complex gut-microbiota—host interactions * Amenable to diet or microbiome-based interventions* Possible long-term experiments *Requires housing facility and adequate agreements* Expensive to maintain colonies*Housing husbandries and diets can modulate mouse microbiota* Murine gut microbiota different from the human gut microbiota* Mucin glycosylation profile of mice different from human intestinal mucins* No reproduction of the full complexity of the human gut microbiota* Limited translational capacity to human situation* Mice generally inbred so no reproduction of the genetic variations found in the human population Velcich et al. 2002; Van der Sluis et al. 2006 Types of models Description Applications Advantages Limitations References In vitro mucus/mucin binding assays Microplates—Flow chambers * Immobilization of mucus/mucin on the microtiter plate* Microtiter plate: adhesion in static conditions* Flow chambers: adhesion under dynamic conditions (fluid shear) * Evaluation of bacterial adhesion (commensals and pathogens) to mucins and molecular mechanisms associated * Fast, quantitative and high throughput method to study mucus-microbe interactions independently from other in vivo conditions* Identification of molecular determinants involved in adhesion of microbes* Coupling with biophysical techniques (Surface Plasmon Resonance, Atomic Force Microscopy) * Influence of experimental conditions (antibiotics, mechanical treatments, growth conditions, hydrophobic interactions)* Limited availability of purified mucins (mainly use of pig gastric mucin)* Absence of gut microbiota McNamara et al. 2000; Gusils et al. 2004; Ringot-Destrez et al.2018; Clyne et al.2017; Dunne et al. 2018 In vitro cell models Monoculture models *Gut–derived epithelial cells resembling intestinal tissue consisting mainly of mature goblet cells that secrete an adherent *Adherence of commensal and pathogenic bacteria to host cells * Effect of commensals/pathogens on host cell mucin synthesis and/or composition of the mucus layer *Reproducible and easily handled in laboratories* Identification of molecular determinants involved in adhesion of microbes and host cell mucin synthesis * Good platform for screening and characterizing probiotic activity *Derived from cancer cells, different from healthy tissue* Not representative of various cell types recovered in mucosal epithelial tissues* Not representative of appropriate MUC gene expression* Modulation of mucus production by culture conditions * Absence of gut microbiota* Difficulty to maintain for long-term experiments (> 1 month)* Requirement of high oxygen levels (difficulty to study oxygen-sensitive microbes) Linden et al. 2007; Navabi et al. 2013; Hews et al. 2017 Co-culture models *Mixed culture of enterocytes and mucin secreting cells *Adherence of commensal and pathogenic bacteria to host cells * Effect of commensals/pathogens on host cell mucin synthesis and/or composition of the mucus layer *Better representation of cell-type ratio recovered in mucosal epithelial tissues* Simple model, well described in literature *Absence of M-cells (development of triple co-culture Caco-2/HT29-MTX/Raji B) * Variations in seeding ratios of HT29 MTX/Caco-2 can impede results interpretation* Modulation of mucus production by culture conditions * Absence of gut microbiota* Difficulty to maintain for long-term experiments (> 1 month)* Requirement of high oxygen levels (difficulty to study oxygen-sensitive microbes) Hilgendorf et al. 2000; Lozoya-Agullo et al. 2017 Ex-vivo organ cultures Intestinal organoids *Generation of self-propagating spheres of primary intestinal epithelial cells * Enteroids = derived from adult stem cells isolated from the crypts of human small intestinal * Colonoids = derived from adult stem cells isolated from the crypts of human colonic tissue *Study of advanced aspects of mucus development in a more complex scenario* Study of host–commensals and pathogens interactions *Often collected from mice tissues, possible use of patient-derived tissues* Assay that more accurately mimics in vivo conditions* Amenable to long-term culture *Highly expensive and requires specialized expertise* Requires access to biopsies/tissues * Donor-to-donor variability* Requirement of injection to infect organoids with bacteria* Absence of gut microbiota* No reproduction of peristalsis motions and GI stressful events* Requirement of high oxygen levels (difficulty to study oxygen-sensitive microbes) Jung et al. 2011; Sato et al. 2011 In vitro organ culture (IVOC) *Whole organs maintained in vitro *Study of host–commensals and pathogens interactions *Better maintenance of tissue architecture *Presence of non-transformed cells including all major cell types (enterocytes, goblet cells, Paneth cells and endocrine cells)* Often collected from animal tissues, possible use of patient-derived tissues* Possible use of biopsies from disease patients (e.g. IBD) *Requires access to biopsies/tissues * Expensive and requires expertise *Donor-to-donor variability* Difficulty to maintain for long-term experiments* No reproduction of peristalsis motions and GI stressful events* Requirement of high oxygen levels (difficulty to study oxygen-sensitive microbes) Browning and Trier 1969; Schüller et al. 2007 Gut- on-a chip *Reproduction of the multicellular structures, cell–cell and tissue–tissue interactions, and the native microenvironment* Closely reproduction of the in vivo situation *Study of the complex physiological and pathophysiological responses of tissues at an organ level* Study of host–commensals and pathogens interactions *Presence of non-transformed cells including all major cell types (enterocytes, goblet cells, Paneth cells and endocrine cells)* Reproduction of peristalsis like motions * Possible use of biopsies from disease patients (e.g. IBD) *Expensive and requires dedicated expertise and instrumentation* Stem cell differentiation is difficult to achieve* Flow rate of the medium can influence cell metabolism* Absence of gut microbiota* No input from immune and nervous system* Requirement of high oxygen levels (difficulty to study oxygen-sensitive microbes)* No reproduction of the full complexity of the human gut microbiota Kasendra et al. 2018 In vitro human colonic models involving a mucosal phase M-SHIME *Series of bioreactor modeling the different parts of the human gut* Introduction of mucus-coated carriers (Mucus SHIME) *Study the fine-scale spatial organization of the gut microbial ecosystem* Investigation of the interactions between commensals, pathogens, probiotics and luminal/mucosal gut microbiota *Integration of human GI -related parameters and possibility to modulate them depending on diet, age and diseases (e.g. ulcerative colitis)* Capture dynamics by time-resolved analyses* Capture inter-individual variability of human gut microbiota* Possible long term experiments* Possible coupling with cell culture models and Host-Microbe Interactions (HMI) module *Expensive and requires expertise and specialized instrumentation* Use of pig gastric mucin* No reproduction of immune and nervous system* No reproduction of the full complexity of the human gut microbiota* Donor-to-donor variability Van den Abbeele et al. 2012; Van den Abbeele et al. 2013; De Paepe et al. 2018 In vivo animal models *Whole organism models* Development of genetically modified mice with impaired mucin production (comparison with wild type animals) *Study of the functional roles of mucin and mucus under physiological or pathological conditions at the level of entire organism* Investigation of downstream consequences of mucin modulation in mucosal barrier defense* Investigation of the interactions between commensals, pathogens, probiotics and luminal/mucosal gut microbiota *Physiological model* Allow targeting of a specific gene/pathway in the complex gut-microbiota—host interactions * Amenable to diet or microbiome-based interventions* Possible long-term experiments *Requires housing facility and adequate agreements* Expensive to maintain colonies*Housing husbandries and diets can modulate mouse microbiota* Murine gut microbiota different from the human gut microbiota* Mucin glycosylation profile of mice different from human intestinal mucins* No reproduction of the full complexity of the human gut microbiota* Limited translational capacity to human situation* Mice generally inbred so no reproduction of the genetic variations found in the human population Velcich et al. 2002; Van der Sluis et al. 2006 Open in new tab Table 1. Experimental models available to study mucus-bacteria interactions.GI : Gastro-Intestinal, HMI : Host–Microbe Interactions, IBD : Inflammatory Bowel Ddiseases, IVOC : In vitro organ culture, M-SHIME : Mucus Simulator of the Human Intestinal Microbial Ecosystem. Types of models Description Applications Advantages Limitations References In vitro mucus/mucin binding assays Microplates—Flow chambers * Immobilization of mucus/mucin on the microtiter plate* Microtiter plate: adhesion in static conditions* Flow chambers: adhesion under dynamic conditions (fluid shear) * Evaluation of bacterial adhesion (commensals and pathogens) to mucins and molecular mechanisms associated * Fast, quantitative and high throughput method to study mucus-microbe interactions independently from other in vivo conditions* Identification of molecular determinants involved in adhesion of microbes* Coupling with biophysical techniques (Surface Plasmon Resonance, Atomic Force Microscopy) * Influence of experimental conditions (antibiotics, mechanical treatments, growth conditions, hydrophobic interactions)* Limited availability of purified mucins (mainly use of pig gastric mucin)* Absence of gut microbiota McNamara et al. 2000; Gusils et al. 2004; Ringot-Destrez et al.2018; Clyne et al.2017; Dunne et al. 2018 In vitro cell models Monoculture models *Gut–derived epithelial cells resembling intestinal tissue consisting mainly of mature goblet cells that secrete an adherent *Adherence of commensal and pathogenic bacteria to host cells * Effect of commensals/pathogens on host cell mucin synthesis and/or composition of the mucus layer *Reproducible and easily handled in laboratories* Identification of molecular determinants involved in adhesion of microbes and host cell mucin synthesis * Good platform for screening and characterizing probiotic activity *Derived from cancer cells, different from healthy tissue* Not representative of various cell types recovered in mucosal epithelial tissues* Not representative of appropriate MUC gene expression* Modulation of mucus production by culture conditions * Absence of gut microbiota* Difficulty to maintain for long-term experiments (> 1 month)* Requirement of high oxygen levels (difficulty to study oxygen-sensitive microbes) Linden et al. 2007; Navabi et al. 2013; Hews et al. 2017 Co-culture models *Mixed culture of enterocytes and mucin secreting cells *Adherence of commensal and pathogenic bacteria to host cells * Effect of commensals/pathogens on host cell mucin synthesis and/or composition of the mucus layer *Better representation of cell-type ratio recovered in mucosal epithelial tissues* Simple model, well described in literature *Absence of M-cells (development of triple co-culture Caco-2/HT29-MTX/Raji B) * Variations in seeding ratios of HT29 MTX/Caco-2 can impede results interpretation* Modulation of mucus production by culture conditions * Absence of gut microbiota* Difficulty to maintain for long-term experiments (> 1 month)* Requirement of high oxygen levels (difficulty to study oxygen-sensitive microbes) Hilgendorf et al. 2000; Lozoya-Agullo et al. 2017 Ex-vivo organ cultures Intestinal organoids *Generation of self-propagating spheres of primary intestinal epithelial cells * Enteroids = derived from adult stem cells isolated from the crypts of human small intestinal * Colonoids = derived from adult stem cells isolated from the crypts of human colonic tissue *Study of advanced aspects of mucus development in a more complex scenario* Study of host–commensals and pathogens interactions *Often collected from mice tissues, possible use of patient-derived tissues* Assay that more accurately mimics in vivo conditions* Amenable to long-term culture *Highly expensive and requires specialized expertise* Requires access to biopsies/tissues * Donor-to-donor variability* Requirement of injection to infect organoids with bacteria* Absence of gut microbiota* No reproduction of peristalsis motions and GI stressful events* Requirement of high oxygen levels (difficulty to study oxygen-sensitive microbes) Jung et al. 2011; Sato et al. 2011 In vitro organ culture (IVOC) *Whole organs maintained in vitro *Study of host–commensals and pathogens interactions *Better maintenance of tissue architecture *Presence of non-transformed cells including all major cell types (enterocytes, goblet cells, Paneth cells and endocrine cells)* Often collected from animal tissues, possible use of patient-derived tissues* Possible use of biopsies from disease patients (e.g. IBD) *Requires access to biopsies/tissues * Expensive and requires expertise *Donor-to-donor variability* Difficulty to maintain for long-term experiments* No reproduction of peristalsis motions and GI stressful events* Requirement of high oxygen levels (difficulty to study oxygen-sensitive microbes) Browning and Trier 1969; Schüller et al. 2007 Gut- on-a chip *Reproduction of the multicellular structures, cell–cell and tissue–tissue interactions, and the native microenvironment* Closely reproduction of the in vivo situation *Study of the complex physiological and pathophysiological responses of tissues at an organ level* Study of host–commensals and pathogens interactions *Presence of non-transformed cells including all major cell types (enterocytes, goblet cells, Paneth cells and endocrine cells)* Reproduction of peristalsis like motions * Possible use of biopsies from disease patients (e.g. IBD) *Expensive and requires dedicated expertise and instrumentation* Stem cell differentiation is difficult to achieve* Flow rate of the medium can influence cell metabolism* Absence of gut microbiota* No input from immune and nervous system* Requirement of high oxygen levels (difficulty to study oxygen-sensitive microbes)* No reproduction of the full complexity of the human gut microbiota Kasendra et al. 2018 In vitro human colonic models involving a mucosal phase M-SHIME *Series of bioreactor modeling the different parts of the human gut* Introduction of mucus-coated carriers (Mucus SHIME) *Study the fine-scale spatial organization of the gut microbial ecosystem* Investigation of the interactions between commensals, pathogens, probiotics and luminal/mucosal gut microbiota *Integration of human GI -related parameters and possibility to modulate them depending on diet, age and diseases (e.g. ulcerative colitis)* Capture dynamics by time-resolved analyses* Capture inter-individual variability of human gut microbiota* Possible long term experiments* Possible coupling with cell culture models and Host-Microbe Interactions (HMI) module *Expensive and requires expertise and specialized instrumentation* Use of pig gastric mucin* No reproduction of immune and nervous system* No reproduction of the full complexity of the human gut microbiota* Donor-to-donor variability Van den Abbeele et al. 2012; Van den Abbeele et al. 2013; De Paepe et al. 2018 In vivo animal models *Whole organism models* Development of genetically modified mice with impaired mucin production (comparison with wild type animals) *Study of the functional roles of mucin and mucus under physiological or pathological conditions at the level of entire organism* Investigation of downstream consequences of mucin modulation in mucosal barrier defense* Investigation of the interactions between commensals, pathogens, probiotics and luminal/mucosal gut microbiota *Physiological model* Allow targeting of a specific gene/pathway in the complex gut-microbiota—host interactions * Amenable to diet or microbiome-based interventions* Possible long-term experiments *Requires housing facility and adequate agreements* Expensive to maintain colonies*Housing husbandries and diets can modulate mouse microbiota* Murine gut microbiota different from the human gut microbiota* Mucin glycosylation profile of mice different from human intestinal mucins* No reproduction of the full complexity of the human gut microbiota* Limited translational capacity to human situation* Mice generally inbred so no reproduction of the genetic variations found in the human population Velcich et al. 2002; Van der Sluis et al. 2006 Types of models Description Applications Advantages Limitations References In vitro mucus/mucin binding assays Microplates—Flow chambers * Immobilization of mucus/mucin on the microtiter plate* Microtiter plate: adhesion in static conditions* Flow chambers: adhesion under dynamic conditions (fluid shear) * Evaluation of bacterial adhesion (commensals and pathogens) to mucins and molecular mechanisms associated * Fast, quantitative and high throughput method to study mucus-microbe interactions independently from other in vivo conditions* Identification of molecular determinants involved in adhesion of microbes* Coupling with biophysical techniques (Surface Plasmon Resonance, Atomic Force Microscopy) * Influence of experimental conditions (antibiotics, mechanical treatments, growth conditions, hydrophobic interactions)* Limited availability of purified mucins (mainly use of pig gastric mucin)* Absence of gut microbiota McNamara et al. 2000; Gusils et al. 2004; Ringot-Destrez et al.2018; Clyne et al.2017; Dunne et al. 2018 In vitro cell models Monoculture models *Gut–derived epithelial cells resembling intestinal tissue consisting mainly of mature goblet cells that secrete an adherent *Adherence of commensal and pathogenic bacteria to host cells * Effect of commensals/pathogens on host cell mucin synthesis and/or composition of the mucus layer *Reproducible and easily handled in laboratories* Identification of molecular determinants involved in adhesion of microbes and host cell mucin synthesis * Good platform for screening and characterizing probiotic activity *Derived from cancer cells, different from healthy tissue* Not representative of various cell types recovered in mucosal epithelial tissues* Not representative of appropriate MUC gene expression* Modulation of mucus production by culture conditions * Absence of gut microbiota* Difficulty to maintain for long-term experiments (> 1 month)* Requirement of high oxygen levels (difficulty to study oxygen-sensitive microbes) Linden et al. 2007; Navabi et al. 2013; Hews et al. 2017 Co-culture models *Mixed culture of enterocytes and mucin secreting cells *Adherence of commensal and pathogenic bacteria to host cells * Effect of commensals/pathogens on host cell mucin synthesis and/or composition of the mucus layer *Better representation of cell-type ratio recovered in mucosal epithelial tissues* Simple model, well described in literature *Absence of M-cells (development of triple co-culture Caco-2/HT29-MTX/Raji B) * Variations in seeding ratios of HT29 MTX/Caco-2 can impede results interpretation* Modulation of mucus production by culture conditions * Absence of gut microbiota* Difficulty to maintain for long-term experiments (> 1 month)* Requirement of high oxygen levels (difficulty to study oxygen-sensitive microbes) Hilgendorf et al. 2000; Lozoya-Agullo et al. 2017 Ex-vivo organ cultures Intestinal organoids *Generation of self-propagating spheres of primary intestinal epithelial cells * Enteroids = derived from adult stem cells isolated from the crypts of human small intestinal * Colonoids = derived from adult stem cells isolated from the crypts of human colonic tissue *Study of advanced aspects of mucus development in a more complex scenario* Study of host–commensals and pathogens interactions *Often collected from mice tissues, possible use of patient-derived tissues* Assay that more accurately mimics in vivo conditions* Amenable to long-term culture *Highly expensive and requires specialized expertise* Requires access to biopsies/tissues * Donor-to-donor variability* Requirement of injection to infect organoids with bacteria* Absence of gut microbiota* No reproduction of peristalsis motions and GI stressful events* Requirement of high oxygen levels (difficulty to study oxygen-sensitive microbes) Jung et al. 2011; Sato et al. 2011 In vitro organ culture (IVOC) *Whole organs maintained in vitro *Study of host–commensals and pathogens interactions *Better maintenance of tissue architecture *Presence of non-transformed cells including all major cell types (enterocytes, goblet cells, Paneth cells and endocrine cells)* Often collected from animal tissues, possible use of patient-derived tissues* Possible use of biopsies from disease patients (e.g. IBD) *Requires access to biopsies/tissues * Expensive and requires expertise *Donor-to-donor variability* Difficulty to maintain for long-term experiments* No reproduction of peristalsis motions and GI stressful events* Requirement of high oxygen levels (difficulty to study oxygen-sensitive microbes) Browning and Trier 1969; Schüller et al. 2007 Gut- on-a chip *Reproduction of the multicellular structures, cell–cell and tissue–tissue interactions, and the native microenvironment* Closely reproduction of the in vivo situation *Study of the complex physiological and pathophysiological responses of tissues at an organ level* Study of host–commensals and pathogens interactions *Presence of non-transformed cells including all major cell types (enterocytes, goblet cells, Paneth cells and endocrine cells)* Reproduction of peristalsis like motions * Possible use of biopsies from disease patients (e.g. IBD) *Expensive and requires dedicated expertise and instrumentation* Stem cell differentiation is difficult to achieve* Flow rate of the medium can influence cell metabolism* Absence of gut microbiota* No input from immune and nervous system* Requirement of high oxygen levels (difficulty to study oxygen-sensitive microbes)* No reproduction of the full complexity of the human gut microbiota Kasendra et al. 2018 In vitro human colonic models involving a mucosal phase M-SHIME *Series of bioreactor modeling the different parts of the human gut* Introduction of mucus-coated carriers (Mucus SHIME) *Study the fine-scale spatial organization of the gut microbial ecosystem* Investigation of the interactions between commensals, pathogens, probiotics and luminal/mucosal gut microbiota *Integration of human GI -related parameters and possibility to modulate them depending on diet, age and diseases (e.g. ulcerative colitis)* Capture dynamics by time-resolved analyses* Capture inter-individual variability of human gut microbiota* Possible long term experiments* Possible coupling with cell culture models and Host-Microbe Interactions (HMI) module *Expensive and requires expertise and specialized instrumentation* Use of pig gastric mucin* No reproduction of immune and nervous system* No reproduction of the full complexity of the human gut microbiota* Donor-to-donor variability Van den Abbeele et al. 2012; Van den Abbeele et al. 2013; De Paepe et al. 2018 In vivo animal models *Whole organism models* Development of genetically modified mice with impaired mucin production (comparison with wild type animals) *Study of the functional roles of mucin and mucus under physiological or pathological conditions at the level of entire organism* Investigation of downstream consequences of mucin modulation in mucosal barrier defense* Investigation of the interactions between commensals, pathogens, probiotics and luminal/mucosal gut microbiota *Physiological model* Allow targeting of a specific gene/pathway in the complex gut-microbiota—host interactions * Amenable to diet or microbiome-based interventions* Possible long-term experiments *Requires housing facility and adequate agreements* Expensive to maintain colonies*Housing husbandries and diets can modulate mouse microbiota* Murine gut microbiota different from the human gut microbiota* Mucin glycosylation profile of mice different from human intestinal mucins* No reproduction of the full complexity of the human gut microbiota* Limited translational capacity to human situation* Mice generally inbred so no reproduction of the genetic variations found in the human population Velcich et al. 2002; Van der Sluis et al. 2006 Open in new tab In vitro mucus/mucin binding assays Microplate assays Several microtiter plate assays have been developed for testing bacterial adhesion to mucus and/or mucin (McNamara, Sack and Fleiszig 2000; Gusils, Morata and Gonzalez 2004). These generally rely on the immobilisation of mucins or mucus to the wells of microtiter plates following incubation overnight at 4°C or at room temperature in buffers such as such as PBS (pH 7–7.5), HBSS (pH 7–7.5) or carbonate buffer (pH 9.6) (Gusils, Morata and Gonzalez 2004; Dague et al. 2010; Mackenzie et al. 2010; Chagnot et al. 2013). BSA (bovine serum albumin) is generally used as a negative control for assessing the specificity of the binding to mucus and/or mucin. Binding assays are usually performed at 37°C and the contact time with bacterial cells generally ranges between 30 min to 3 hours before washing to remove non-adhered bacteria. Antibiotic at growth inhibiting concentration, such as chloramphenicol, or sometimes thermic treatment can be applied to prevent the growth of microorganisms in the course of the adhesion assay. Binding can be determined using crystal violet staining of the adhered microbial biomass (Azeredo et al. 2017) or by Enzyme-linked Immunosorbent Assay (ELISA) when specific antibodies against bacteria are available (Skoog et al. 2012), by measurement of viable counts after plating of the cells (McNamara, Sack and Fleiszig 2000) or by quantitative PCR (Skoog et al. 2012). Alternatively, bacteria can be labelled with a radioactive probe or a fluorescent dye before inoculation and the binding quantified using a scintillation counter or a fluorometer, respectively (Gusils, Morata and Gonzalez 2004; Mackenzie et al. 2010). Microbial cells can also be labelled by biotinylation and further assayed using streptavidin-HRP by ELISA (Sheng et al. 2012). Quantification of microbial binding to mucin can also be performed by flow cytometry, where microbial cells are put in contact with mucin labelled with a fluorescent tag (de Repentigny et al. 2000). Dot blot assay More recently, a dot-blot method was developed for the sensitive and rapid detection of microorganisms able to bind to mucins (Ringot-Destrez et al. 2018). In brief, purified mucins were spotted on a nitrocellulose membrane, whereas the bacterial cells were labelled using a fluorescent dye, such as 4',6-diamidino-2-phenylindole (DAPI), Syto9 or Fluorescein isothiocyanate (FITC), before being overlaid (Ringot-Destrez et al. 2018). The adhesion capacities of the microorganisms tested differed depending on the nature of the mucins including purified GI tract mucins, PGM and mucins from the mucus-secreting cell line such as HT29-MTX (see detailed description of this cell line in Section 4.2) (Ringot-Destrez et al. 2018). Mucin microarrays The carbohydrate microarray technology offers a powerful platform where natural or synthetic glycans are immobilized onto a solid support. (Poole et al. 2018). Microarrays incorporating mucins from various sources onto different chips surfaces provide a high-throughput approach to screen bacteria-mucin interactions as well as identify glycan-binding proteins and glycan epitopes involved in this interaction (Clyne et al. 2017). For example, the use of mucin microarrays revealed that C. jejuni and H. pylori recognised distinct mucin receptors despite being closely related phylogenetically (Naughton et al. 2013). Recently, H. pylori was shown to interact with trefoil factor family (TFF) protein TFF1 (Reeves et al. 2008), and that TFF1 specifically interacts with human gastric mucin but not with human colonic mucins nor mucins from other animal sources as shown using mucin microarrays (Dunne et al. 2018). This indicates that TFF1 may play an important role in the development of gastric cancer in H. pylori infections (Reeves et al. 2008; Dunne et al. 2018). Mucin microarrays were also used to identify the interactions of commensal strains with mucus (Lactobacillus salivarius AH102 and Bifidobacteria longum AH1205), highlighting the importance of mucin glycans in the preference of the two bacteria to mucins (Naughton et al. 2013; Flannery et al. 2015). Binding assays in flow chamber As a consequence of fluid shear gradient in the gut, the bacteria located in the outer mucus layer are exposed to a more turbulent flow compared to those that reside between the microvilli of the epithelial cells and therefore less exposed to physical perturbation (De Weirdt and Van de Wiele 2015). While the assays described above correspond to adhesion under static conditions, experiments can also be performed in dynamic conditions using flow chamber, where the shear force can be controlled (Le et al. 2013). Low-fluid shear environments and high shear rates are known to provide laminar pattern. Over time and with different laminar flow rates, the surface coverage of microbial cells to coupons coated with mucin provides an estimate detachment profile as a function of the shear stress. Biophysical assays In order to gain further molecular insights into the interactions of microbial cells with mucin, various biophysical techniques have been developed and applied over the years. Optical biosensors based on resonant mirrors have been used to determine the binding kinetics of H. pylori cells to mucin (Hirmo et al. 1999). Following competition binding assays, the recognition of sialylated and sulphated moieties of mucin by H. pylori was demonstrated. Surface plasmon resonance (SPR) has been used to evaluate the adhesion abilities of a range of Lactobacillus species (Uchida et al. 2004; Kinoshita et al. 2007). In these studies, human colonic mucin (HCM) was immobilised on the sensor chip whereas bacterial cells were eluted as analytes. Using sialidase or sulfatase, it was further possible to discriminate some strains of lactobacilli and bifidobacteria that could specifically bind to the sialic acid or sulphate residues of HCM respectively (Huang et al. 2013). Single-cell force spectroscopy (SFCS) has been used to quantify the adhesion forces of L. rhamnosus with mucin at a single-cell level, pinpointing heterogeneities in the bacterial population (Sullan et al. 2014). More recently, further molecular details of mucin-bacteria interactions were investigated using atomic force microscopy (AFM). Such an approach was used for the first time to accurately quantify the force of adhesion of L. lactis cells immobilised on the AFM tip to PGM at nanoscale level (Dague et al. 2010). Surprisingly, it was found that PGM coating strongly reduced the bacterial adhesion force compared to bare polystyrene, highlighting the interplay between electrostatic, hydrophilic and steric repulsions, and that both specific and non-specific interactions need to be considered (Dague et al. 2010). These results were consistent with a previous investigation of the muco-adhesive properties of L. lactis using quartz crystal microbalance with dissipation monitoring (Le et al. 2012). Using bacteria mutant strains, AFM was also used to provide molecular insights into the respective role and contribution of mucus-binding proteins and surface organelles (pili or flagella) in muco-adhesion (Le et al. 2013). Interactions at the protein-protein level were further investigated by AFM to study the adhesive properties of L. reuteri Mub with mucins (Gunning et al. 2016). In vitro mucin-secreting cell models Monoculture models While many colon carcinoma cell lines express mRNAs encoding surface-associated and/or secreted intestinal mucins (Deplancke and Gaskins 2001), few of them secrete MUC2 or form a mucus layer (Linden, Driessen and McGuckin 2007; Navabi, McGuckin and Lindén 2013; Hews et al. 2017). Most mucus-secreting cell lines are derived from the heterogeneous adenocarcinoma cell line HT-29 which can be differentiated into a mucus-secreting phenotype by growth under metabolic stress conditions. After an initial phase of cell mortality, adapted subpopulations of highly differentiated cells emerge (Lievin-Le Moal and Servin 2013). HT29–18N2 cells are often used as a model system for goblet cell differentiation and mucin secretion; these cells have been established by growth under glucose deprivation in galactose-containing culture medium (Phillips et al. 1988). In contrast, HT29-MTX cells and their clonal derivatives have been obtained by sequential adaptation to increasing concentrations of methotrexate (Lesuffleur et al. 1990). When grown on Transwell filter supports, some HT-29 MTX clones (e.g. MTX-D1 and MTX-E12) form polarised monolayers mostly constituted of mature goblet cells secreting an adherent mucus layer of 50–150 µm thickness as revealed by Alcian Blue staining (Behrens et al. 2001). In addition, the mucin-secreting clonal cell line HT-29.cl16E emerged from parental HT-29 cells after subculture in sodium butyrate whilst HT29-FU cells were established by treatment with 5-fluorouracil (Lesuffleur et al. 1991). These mucus-producing HT-29 derivatives have been widely used to investigate the adherence of commensal and pathogenic bacteria to host cells (Coconnier et al. 1992; Bernet et al. 1993; Eveillard et al. 1993; Bernet et al. 1994; Kerneis et al. 1994; Favre-Bonte, Joly and Forestier 1999; Gopal et al. 2001; Schild et al. 2005; Barketi-Klai et al. 2011; Dolan et al. 2012; Gagnon et al. 2013; Naughton et al. 2013; Martins et al. 2015; Martins et al. 2016) and/or evaluate the effect of commensal bacteria on infection with enteropathogens (Bernet et al. 1993; Bernet et al. 1994; Coconnier et al. 1998; Gopal et al. 2001; Alemka et al. 2010; Zihler et al. 2011; Zivkovic et al. 2015; Vazquez-Gutierrez et al. 2016). Some studies investigated the direct effect of commensal or pathogenic bacteria on host cell mucin synthesis and/or composition of the mucus layer. Infection with atypical EPEC increased expression of secreted MUC2 and MUC5AC as well as membrane-bound MUC3 and MUC4 in HT29-MTX cells, thereby enhancing bacterial growth by providing nutrients for adherent bacteria (Vieira et al. 2010). Another study showed that apical infection with Listeria monocytogenes stimulated mucus secretion by polarised HT29-MTX cells. This effect was mediated by binding of the toxin listeriolysin O to a receptor on the epithelial brush border (Coconnier et al. 1998) and reduced bacterial invasion and colonisation of the host epithelium (Lievin-Le Moal, Servin and Coconnier-Polter 2005). Interestingly, probiotic Lactobacillus strains which adhering to mucus-producing HT-29 cells upregulated the transcription and secretion of MUC3 which reduced adherence of EPEC in co-incubation experiments (Mack et al. 2003). Modulation of mucus production and mucin glycosylation by commensal bacteria can also occur independently of adhesion. For example, a small soluble peptide of the gut commensal R. gnavus E1 strain has been shown to increase HT-29 MTX cell glycosylation via enhanced transcription of glycosyltransferases and MUC2-encoding genes (Graziani et al. 2016). Similarly, a soluble low molecular weight compound from B. thetaiotaomicron has been reported to enhance galactosylation in HT29-MTX cells. While no change in transcription was detected, galactosyltransferase activity was increased in HT29-MTX cells treated with soluble bacterial extract suggesting post-translational mechanisms of regulation (Miguel et al. 2001). In addition to HT-29 cell derivatives, mucus-producing LS174T colon carcinoma cells have been used to study host-bacteria interactions. LS174T cells secrete mature MUC2, MUC5AC and human gallbladder mucin (van Klinken et al. 1996) but do not produce an organised adherent mucus layer (Navabi, McGuckin and Lindén 2013). Recent studies using this cell line showed that the secreted metalloprotease StcE reduced MUC2 levels during infection with EHEC and thereby facilitated bacterial adherence to the intestinal epithelium (Hews et al. 2017). In addition, the soluble protein p40 from L. rhamnosus GG stimulated MUC2 mRNA and protein expression in LS174T cells, and this effect was dependent on the epidermal growth factor receptor (Wang et al. 2014). Furthermore, treatment with butyrate, a product of bacteria fermentation, increased mucin production in LS174T cells (Burger-van Paassen et al. 2009; Jung et al. 2015). Recently, LS174T cells were used to decipher E. histolytica-elicited suppressed goblet cell transcription (Leon-Coria et al. 2018). Co-culture models To model human intestinal epithelia, mixed cultures of enterocyte-like Caco-2 cells and mucus-producing HT29-MTX cells have been widely used in drug absorption and permeability studies (Hilgendorf et al. 2000; Lozoya-Agullo et al. 2017). Co-cultures prepared with different ratios of Caco-2 and HT29-MTX cells seeded out on Transwell inserts formed a continuous mucus layer similar to cultures of HT-29-MTX cells grown alone (Poquet, Clifford and Williamson 2008; Beduneau et al. 2014). Notably, the probiotic strains L. rhamnosus GG or bifidobacteria as well as pathogenic strains of E. coli or L. monocytogenes adhered better to mucus-deficient Caco-2 cells than to mucus-producing HT-29MTX cells or Caco-2/HT-29 co-cultures (Laparra and Sanz 2009). Considering the ‘closed’ oxygen-restricted environment in the human gut, Chen and colleagues developed a 3D porous silk scaffolding in the shape of a hollow tube. While the inner tube wall was coated with Caco-2/HT29-MTX epithelia, primary human intestinal myofibroblasts were grown in the tube scaffold space underneath to support epithelial growth and differentiation. Notably, epithelia grown on 3D scaffolds demonstrated increased MUC2 production compared to Transwell cultures resulting in the formation of a mucus layer of 11–17 μm thickness (Chen et al. 2015). Organ-on-a-chip Another approach to simulate a mucin-producing human intestinal epithelium is the ‘Gut-on-a-Chip’ system, where Caco-2 cells are grown on a porous membrane support in a microfluidic device. While the cell membrane support is maintained under cyclic strain mimicking peristaltic motion, the chambers above and below the cell membrane are constantly perfused with medium, thereby generating low shear stress. This environment stimulates the formation of 3D intestinal villi similar to those found in the small intestine (Kim et al. 2012), and the differentiation of Caco-2 cell into absorptive enterocytes, and also includes enteroendocrine cells, Paneth cells and mucus-producing goblet cells (Kim and Ingber 2013). In addition, Caco-2 epithelium grown in the Gut-on-a-Chip model display enhanced barrier function and mucus production as compared to static Caco-2 cell cultures (Kim and Ingber 2013). This system has recently been developed further to allow co-culture with strict anaerobes (Shin et al. 2019). Although the Gut-on-a-Chip devices have been mostly used for long-term co-culture of IECs with commensal microbes under healthy conditions, they are now being employed to model intestinal inflammation (Kim et al. 2016). Using a Gut-on-a-Chip model, the pathophysiological manifestation and dysregulated barrier function observed during inflammation could be recapitulated which may help to gain insights into disease mechanisms and assess potential therapeutic strategies (Shin and Kim 2018). Notably, probiotic VSL#3 targeted restoration of the mucosal barrier did not effectively control the local inflammation nor improve mucus production (Shin and Kim 2018). The HuMix (Human Microbial Cross-talk) model is another microfluidic device enabling the co-culture of Caco-2 cell monolayers with commensal bacteria under anaerobic conditions. In contrast to the Gut-on-a-Chip system, the epithelial cells which do not produce mucus are separated from the bacteria by a membrane coated with porcine gastric mucin (Shah et al. 2016). Ex vivo organ cultures As described above, traditional culture of human cells represents a valuable predictor of human physiology, pathology, and therapeutic responses but is limited by the absence of the tissue microenvironment. Culture approaches using human intestinal biopsy samples therefore represent an upscale platform to investigate the involvement of the mucus layer in healthy conditions or in the onset of various diseases. In vitro organ culture (IVOC) In 1969, Browning and Trier were the first to establish a technique to culture human mucosal biopsies ex vivo. By using a specific culture medium and incubation of the samples in 95% O2, 5% CO2 at 37°C, mucosal biopsies from the duodeno-jejunal junction were kept alive for up to 24 hours demonstrating epithelial cell proliferation, fat absorption and active mucus secretion by goblet cells (Browning and Trier 1969). The advantages of IVOC of intestinal biopsies versus cell line culture models include the presence of healthy non-transformed cells including all major IEC types (enterocytes, goblet cells, Paneth cells and neuroendocrine cells), underlying basement membrane and mucosal tissue, and the production of mucus. While it is problematic to maintain the loose outer mucus layer of colonic biopsies during sampling, the inner colonic and small intestinal mucus layers are generally well preserved as evidenced by microscopy (Haque et al. 2004; Walsham et al. 2016; Hews et al. 2017). IVOC of biopsy samples has been used to investigate adherence of pathogenic bacteria such as EPEC (Knutton, Lloyd and McNeish 1987; Schüller et al. 2007), EHEC (Phillips et al. 2000; Fitzhenry et al. 2002; Lewis et al. 2015), ETEC (Knutton et al. 1989; Baker, Moxley and Francis 1997) and C. jejuni (Grant, Woodward and Maskell 2006) to human intestinal mucosa. In addition, IVOC demonstrated cytotoxic effects of bacterial toxins, such as Pet toxin from EAEC (Henderson et al. 1999b), Shiga toxin from EHEC (Schüller, Frankel and Phillips 2004) and C. difficile toxin A (Mahida et al. 1996) on intestinal epithelium or mucosa. Interactions of enteropathogenic bacteria with mucus production were observed in small intestinal and colonic biopsy tissue infected with EAEC where bacteria were predominantly associated with a thick mucus layer above the epithelium, which was not present in non-infected control samples (Hicks, Candy and Phillips 1996; Andrade, Freymuller and Fagundes-Neto 2011). This suggests that EAEC stimulates mucus secretion which agrees with the production of mucoid stools during EAEC diarrhoea (Croxen et al. 2013). Similarly, stimulation of mucus secretion and bacterial binding to the mucus layer were observed in biopsy samples from the terminal ileum infected with S. Typhimurium. This was followed by Salmonella adherence and invasion of the epithelium accompanied by ruffling of the host cell membrane (Haque et al. 2004). Recently, the IVOC system was used to show that the metalloprotease StcE diminishes the inner mucus layer and enhances EHEC adherence to human colonic biopsy epithelium (Hews et al. 2017). Polarised IVOC (pIVOC) While the traditional IVOC system allows bacterial access to the mucosal and submucosal side of the biopsy, polarised organ culture models have been developed which limit bacterial contact to the mucosal side of the tissue. This is particularly relevant when studying host responses to bacterial infections where artificial interactions with immune cells in the lamina propria might confound experimental readouts. Using a pIVOC approach by mounting colonic tissue explants between two Perspex disks in a Snapwell plate, Raffatellu and colleagues demonstrated that Salmonella Typhi reduced mucosal expression of the pro-inflammatory cytokine interleukin (IL)-8 by production of a capsule which masked pathogen-associated molecular patterns such as LPS and flagellin (Raffatellu et al. 2005). In addition, pIVOC showed that apical exposure to EPEC or purified H6 flagellin induced IL-8 expression in duodenal biopsies (Schüller et al. 2009). Furthermore, infection with C. jejuni stimulated the production of reactive oxygen species (ROS) in duodenal and colonic mucosa (Corcionivoschi et al. 2012). The pIVOC system has also been used to study the interaction of probiotic bacteria with mucosal tissue, and incubation of duodenal explants with L. reuteri demonstrated localisation of bacteria in the mucus layer but not in the epithelium. Nevertheless, pre-incubation with L. reuteri reduced EPEC adherence to the epithelium (Walsham et al. 2016). A different approach to restrict bacterial access to the epithelial surface was developed by Tsilingiri and colleagues by gluing a perspex cylinder to the mucosal side of colonic resection tissue (Tsilingiri et al. 2012). Surprisingly, apical incubation with probiotic L. plantarum resulted in degeneration of mucosal tissue from healthy donors, whilst all three strains studied (L. paracasei, L. rhamnosus, L. plantarum) caused tissue damage in resections from patients with IBD. In contrast, supernatants from L. paracasei reduced inflammation in Salmonella-infected and IBD tissue. As the maintenance of larger tissue samples requires incubation in high levels of oxygen (95–99%), the use of IVOC to study interactions of oxygen-sensitive bacteria with human intestinal mucosa remains problematic. However, a novel murine 3D-intestinal organ culture system was recently developed whereby an intact intestinal fragment was luminally perfused with de-gassed medium containing anaerobic bacteria while the serosal side of the tissue was maintained under humidified oxygenated conditions. Whilst preserving gut tissue architecture, the system also supported the growth of commensal microbes (Clostridium ramosum and SFB) and allowed assessment of their impact on the immune and nervous system (Yissachar et al. 2017). Human enteroids/colonoids and intestinal organoids New technologies have been developed which enable the generation of self-propagating spheres of primary intestinal epithelial cells (‘mini-guts’). Enteroids or colonoids are derived from adult stem cells isolated from the crypts of human small intestinal or colonic tissue, respectively (Jung et al. 2011; Sato et al. 2011). In contrast, human intestinal organoids (HIOs) are established by differentiation of embryonic or, more often, induced pluripotent stem cells (genetically reprogrammed adult stem cells) (Spence et al. 2011). In comparison to enteroids, HIOs lack maturation and more closely resemble foetal than adult intestine. In addition, they are devoid of functional intestinal stem cells and surrounded by a mesenchyme which is absent in enteroids (Sinagoga and Wells 2015; Leslie and Young 2016). As the apical side of the epithelium is facing inwards, infection of spheroid enteroids/HIOs with bacteria requires microinjection. Studies on the anaerobic pathogen C. difficile showed that injected bacteria remained alive in HIOs for up to 12 hours and caused disruption of epithelial barrier function via secretion of the toxin TcdA. Interestingly, oxygen measurements indicated reduced oxygen levels in the lumen of HIOs (5 to 15%). Furthermore, infection with C. difficile resulted in reduced MUC2 and mucus production in HIOs (Engevik et al. 2015). HIOs also supported growth of EHEC and commensal E. coli. Infection with EHEC induced ROS production and an inflammatory response associated with recruitment of external neutrophils into HIO spheres (Karve et al. 2017). Interestingly, colonisation of HIOs with commensal E. coli (ECOR2) stimulated enterocyte maturation, antimicrobial peptide secretion, production of a MUC2-containing mucus layer and increased epithelial barrier function, thereby indicating the establishment of stable host–microbe symbiosis (Hill et al. 2017). To facilitate incubations with bacteria, 2D enteroid systems have now been successfully developed where primary intestinal cells are grown as monolayers on permeable membrane supports. Previous studies showed that differentiated human enteroid and colonoid monolayers contained MUC2-producing goblet cells and formed a mucus layer of more than 25 μm thickness (VanDussen et al. 2015; In et al. 2016). Two-dimensional enteroids and colonoids supported binding of EAEC, EHEC and EPEC (VanDussen et al. 2015). More specifically, apical EHEC infection of colonoids resulted in the formation of characteristic attaching and effacing lesions, mucus degradation and reduced expression of the microvillar protein protocadherin 24, which was mediated by the secreted serine protease EspP (In et al. 2016). The 2D enteroid model was further refined by adding primary human macrophages to the basolateral side of the membrane support. Intriguingly, enteroid monolayers grown in the presence of macrophages exhibited increased cell height and barrier function. In addition, underlying macrophages were able to capture and kill EPEC and ETEC by extending projections across the epithelial monolayer (Noel et al. 2017). In another approach to mimic the gut environment more closely, cells from human small intestinal enteroids were seeded on tubular silk sponge scaffolds and supported by primary human intestinal myofibroblasts as described for Caco-2/HT29-MTX (Section 4.2). The resulting intestinal model epithelium contained all four major epithelial cell types and exhibited tight junction formation, microvillus polarisation, digestive enzyme secretion and low oxygen tension in the lumen. Moreover, infection with a laboratory strain of E. coli resulted in a significant innate immune response (Chen et al. 2017). Recently, a Gut-on-a-Chip model based on primary intestinal epithelial cells has been developed which also includes co-culture of an underlying endothelium. Human enteroids are cultured on a side of a porous membrane within a microfluidic device whereas the intestinal microvascular endothelium is established on the other side of the filter. This device reproduces the epithelial cells proliferation and host defenses more accurately (Kasendra et al. 2018). Kim and colleagues showed that a human Gut-on-a-Chip micro device colonized by non-pathogenic bacteria (commensal and probiotic bacteria) was able to induce production of a key set of pro-inflammatory cytokines. This device enabled high level of mucus production on micro engineered intestinal villi, therefore providing a protective barrier to maintain long-term stable host–microbe coexistence (Kim et al. 2016). In vitro human fermentation models involving a mucosal phase As aforementioned, the spatial positioning of gut microorganisms in the mucus layer is important with respect to their functional role in the human gut ecosystem. The microbial community residing in the mucus layer across the length of the GI tract is, however, hard to study given the difficulty to sample this region in vivo, especially in human (Macfarlane, McBain and Macfarlane 1997; Flint et al. 2012; Donaldson, Lee and Mazmanian 2016). In vitro colonic models involving a mucosal phase are a valuable alternative to study the fine-scale spatial organisation of the gut microbial ecosystem. Multiple colon in vitro models have been developed over the years, ranging from simple, single stage batch incubations to more complex and representative three stage continuous and semi-continuous reactor models (Miller and Wolin 1981; Gibson, Cummings and Macfarlane 1988; Allison, McFarlan and MacFarlane 1989; Blanquet-Diot et al. 2012; McDonald et al. 2013; Van den Abbeele et al. 2013). These continuous fermentation models, inoculated with faecal samples of donors, recapitulate the main biotic and abiotic parameters of the human colon, such as temperature, pH, residence time, supply of nutritive medium reproducing the composition of ileal effluents, therefore enabling the study of a complex and metabolically active gut microbiota under anaerobiosis conditions. In these fermentation models, the bioreactors can be inoculated with fresh or frozen stools provided by individual or pool of different donors being healthy human volunteers with no history of antibiotic treatment 2 to 6 months before the beginning of the study. Introduction of mucus carriers in human in vitro colonic models Mucins in solution have been frequently included in these colonic models to provide a nutrient source to the gut bacteria, but the study of mucus colonisation by gut bacteria in these systems has been revolutionized by the ability to simulate the viscoelastic gel-like nature of the mucus layer through the incorporation of mucus carriers (Gibson, Cummings and Macfarlane 1988; Macfarlane, Hay and Gibson 1989; Macfarlane, Woodmansey and Macfarlane 2005; Van Herreweghen et al. 2017). MacFarlane first demonstrated a rapid colonisation of an agar-mucus layer during a 48-hour incubation in a two-stage continuous fermentor system by a mixture of Bacteroides, enterobacteria and facultative anaerobes (Macfarlane, Woodmansey and Macfarlane 2005). However, the use of glass tubes, containing this agar-mucus layer, in this set-up did not permit a practical long-term implementation (Van den Abbeele et al. 2009). Mucus-coated beads (mixture of 5% porcine mucin type II and 1% agar) have since been identified as crucial platforms in sustaining microbial diversity by selectively enriching species, which are not thriving in the luminal environment. This mucus interphase was introduced in the Simulator of the Human Intestinal Microbial Ecosystem (SHIME) resulting in the M-SHIME (Mucus SHIME) configuration (Van den Abbeele et al. 2012). This system was first used to assess the fitness of potential probiotic lactobacilli, revealing a pronounced enrichment of Lactobacillus mucosae and L. rhamnosus GG in the mucus beads compared to the luminal environment, contributing to their long-term persistence in M-SHIME (Van den Abbeele et al. 2012). The use of next-generation 16S rRNA gene amplicon sequencing methods and the incorporation of mucus beads in both proximal and distal colon conditions in the M-SHIME, further resulted in the detection of several additional mucus-associated species (De Paepe et al. 2018). Besides Roseburia faecis/Enterococcus rectale, a strong enrichment of Ruminococcus inulinivorans, Clostridium, Bilophila, Anaeroglobus and Veillonellaceae species was observed in the proximal mucus layer compared to the lumen (De Paepe et al. 2018). A. muciniphila, Cloacibacillus evryensis, Pyramidobacter piscolens, Eubacterium contortum and species belonging to Odoribacter, Enterobacteriaceae and Desulfovibrio were predominantly residing in the distal mucus layer (De Paepe et al. 2018). It can be expected that the list of species inhabiting the mucus layer will continue to expand in the future M-SHIME experiments, by continuing to explore the inter-individual variability of faecal samples. Another major advantage of in vitro fermentation models is the possibility to capture dynamics by time-resolved analyses. To minimize disturbance of the system during such analyses, the M-SHIME system was adapted to facilitate a rapid, anaerobic, frequent sampling by mounting sampling ports with an airlock system on top of the SHIME lids. These sampling ports moreover enable the anaerobic addition, transfer and sampling of any insoluble dietary substrate. The adapted model was correspondingly termed Dietary Particle-Mucosal-Simulator of the Human Intestinal Microbial Ecosystem (DP-M-SHIME). The DP-M-SHIME offers a closer proxy for the diversity of in vivo GI microenvironments, as aside from the mucus layer, insoluble dietary particles present an interesting additional niche for microorganisms to physically interact with and colonise (De Paepe et al. 2018). In addition, these colonic in vitro-fermentation models enable the study of severe gut microbiome perturbations such as antibiotic therapy or pathogen invasion, which cannot be performed in humans for obvious ethical reasons. Using M-SHIME, the mucus bead carriers were shown to confer resilience to lactobacilli species against a combined treatment of antibiotics including tetracycline, amoxicillin and ciprofloxacin (Van den Abbeele et al. 2012). In addition, the M-SHIME model has been used to demonstrate the antagonistic effects of probiotics and prebiotics such as L. reuteri, long-chain arabinoxylans or inulin towards AIEC colonisation in the mucosal environment (Van den Abbeele et al. 2016). Another study reported the importance of mucus for providing a protective environment to beneficial gut microbes, such as L. reuteri, to help them escape from stress induced by high loads of linoleic acid, the most common polyunsaturated fatty acid in a WSD (De Weirdt et al. 2013). The M-SHIME model has also been shown to preserve disease signatures, as illustrated by a reduced luminal and mucosal Clostridium cluster XIVa colonisation in colon vessels inoculated with the faecal material of ulcerative colitis patients compared to healthy donors (Vermeiren et al. 2012). The mucin-beads technology has recently been transferred into the Artificial Colon (ARCOL), with adaptations to avoid flushing the bioreactor with nitrogen when mucin-coated beads are being renewed (Cordonnier et al. 2015; Thevenot et al. 2015). Combining colon fermentation models with in vitro cell culture Recent advancements in the field of in vitro fermentation involve the combination of gut microbiota models with a host compartment to assess host–microbiota interactions (Bahrami et al. 2011; Marzorati et al. 2014; Defois et al. 2018). The three-way interactions between host, microbiome and dietary interventions can be examined by applying the supernatants of colonic samples onto human cell lines or combinations thereof, such as co-cultures of enterocytes and immune cells, in Transwell systems (Marzorati et al. 2014). Cytokine and TNF-α production are followed up as markers of intestinal inflammation, whereas the trans-epithelial electrical resistance (TEER) and Lucifer yellow translocation give an indication of epithelial barrier function (Geirnaert et al. 2017). While typically, in human cell line experiments, the short-term effects of a single treatment application onto differentiated cells with a disrupted barrier are being evaluated, recently, a method was developed to test the effects of a probiotic treatment on the development of epithelial barrier integrity during cell differentiation, which is more representative of the in vivo situation (Geirnaert et al. 2017). In order to further improve the in vivo relevance, a host module, such as the Host Microbiota Interaction (HMI) Module (Marzorati et al. 2014), can be coupled to the colonic in vitro fermentation systems described above to directly and continuously recirculate the colonic microbial suspension over a mucus layer that is in indirect contact with enterocytes and/or immune cells. In vivo animal models As described above, in vitro mucin-secreting cell cultures, ex-vivo organ cultures as well as in vitro fermentation models have yielded fundamental insights into the role of mucins and mucus in bacterial interactions with the host. However, the use of in vivo models is necessary to study the biological roles of mucins under physiological or pathological conditions at the level of entire organism. Genetically modified mouse models with an impaired mucin production or glycosylation have been developed to assess the role of mucus in the interaction between gut bacteria and the host in vivo. Muc2−/− mouse model Many in vivo animal studies investigating the role of mucus in gut homeostasis have relied on the use of Muc2−/− mice, lacking the major intestinal mucin Muc2. The first studies based on Muc2−/− mice showed that these animals displayed an impaired epithelial barrier function characterised by aberrant intestinal crypt morphology and altered cell maturation and migration, and that the mice frequently developed adenomas in the small intestine, as well as rectal tumours (Velcich et al. 2002). The microscopic analysis of the colon indicated mucosal thickening, increased proliferation and superficial erosions (Van der Sluis et al. 2006). The development of spontaneous colitis in Muc2 deficient mice indicated that Muc2 is critical for colonic protection (Van der Sluis et al. 2006). A gut microbiota dysbiosis was also observed in the Muc2−/− mice which harboured a pro-inflammatory-like microbiota profile, characterized by an increase in Clostridiales and a decrease in Lactobacillaceae (Huang et al. 2015). Furthermore, it was shown that the spatial compartmentalization of bacteria in the intestine of Muc2−/− mice was compromised and transcriptomic analysis revealed a downregulation of TLR, immune and chemokine signaling pathways compared to wild type mice (Sovran et al. 2015) Also, the expression of the network of IL-22-regulated defense genes was increased in Muc2−/− mice (Sovran et al. 2015). Recent work also confirmed a clear shift in the microbiota composition of Muc2−/− mice, with the Firmicutes phylum enriched and the Bacteroidetes phylum decreased, as well as an increase in genera considered as potential pathogens also (Wu et al. 2018). Muc2−/− mice have been used to test the effect of the probiotic mixture VSL#3 on colonic inflammation and intestinal barrier function (Kumar et al. 2017). This probiotic mixture contains eight strains belonging to Lactobacillus, Bifidobacterium and Streptococcus genera which are usually found in the human intestinal microbiota. In Muc2−/− mice, VSL#3 reduced basal colonic proinflammatory cytokine levels and improved epithelial barrier function. In addition, VSL#3 reduced the level of proinflammatory chemokines and upregulated tissue regeneration growth factors leading to a faster resolution of colitis symptoms in Muc2−/− mice with DSS-induced colitis. This was associated with the restoration of antimicrobial peptide gene expression in the small intestine, and an increased abundance of commensal bacteria in the gut. The authors proposed that these beneficial effects were mediated by acetate, produced by the gut bacteria (Kumar et al. 2017). Treatment of Muc2−/− mice with Lactobacillus spp. could ameliorate spontaneous colitis and led to an increased production of SCFA (Morampudi et al. 2016). Muc2−/− mice have also been used to investigate the role of this mucin to prevent bacterial and parasite infection. Upon infection with C. rodentium, a murine pathogen related to diarrhoeagenic attaching-effacing E. coli, Muc2−/− mice exhibited a rapid weight loss and up to 90% mortality (Bergstrom et al. 2010). Mucin secretion was increased in wild type mice during infection as compared to the uninfected controls, suggesting that mucin production is critical to clear the mucosal surface from pathogenic bacteria. In Muc2−/− mice, commensal bacteria were also found to interact with C. rodentium and host tissues, indicating that Muc2 regulates all forms of intestinal microbiota at the gut surface (Bergstrom et al. 2010). When Muc2−/− mice were infected with Salmonella, they showed a dramatic susceptibility to infection, carrying significantly higher caecal and liver pathogen burdens, and developing significantly higher barrier disruption and higher mortality rates than wild type mice (Zarepour et al. 2013). Colonisation of Muc2−/− mice by enterotoxigenic B. fragilis, a causative agent of acute diarrhoea in humans, led to lethal disease (Hecht et al. 2017). The protective function of Muc2 was also demonstrated in models of T. muris parasitic infection (Hasnain et al. 2010). T. muris is a murine infecting nematode which is used as model of T. trichiura infection in humans, a threat in developing countries. After infection, Muc2−/− mice showed a delayed expulsion of the worms from the intestine compared to wild type mice. In addition, an increase in Muc2 production, observed exclusively in resistant mice, correlated with worm expulsion. The nematodes demonstrated a decrease in their energy status in wild type mice compared to the susceptible Muc2−/− mice (Hasnain et al. 2010). E. histolytica is a human parasite infecting the colon and responsible of amoebic dysentery and/or liver abscesses. E. histolytica specifically colonises the mucus layer by adhering to galactose and GalNAc residues present in Muc2 (Kissoon-Singh et al. 2013). The parasite also induces potent hypersecretion from goblet cells. Kissoon-Singh and colleagues showed that E. histolytica induced a pronounced time-dependent secretory exudate with increased gross pathology scores and serum albumin leakage in Muc2−/− mice. Colonic pathology, secretory responses and increased pro-inflammatory cytokine secretions were also correlated with altered expression of tight junction proteins (Kissoon-Singh et al. 2013). These results demonstrate that colonic mucins confer both luminal and epithelial barrier functions and that, in the absence of Muc2, mice are more susceptible to E. histolytica-induced secretory and pro-inflammatory responses. A recent study using antibiotic treated Muc2−/− and Muc2+/+ littermates showed that E. histolytica elicited robust mucus and water secretions, enhanced pro-inflammatory cytokines and chemokine expression and higher pathology scores as compared to the modest response observed in non-antibiotic treated littermates. Host responses were microbiota specific as mucus secretion and pro-inflammatory responses were attenuated following homologous faecal microbial transplants in antibiotic-treated Muc2+/+ quantified by secretion of 3H-glucosamine newly synthesized mucin, Muc2 mucin immunostaining and immunohistochemistry (Leon-Coria et al. 2018). The mechanism controlling mucus release in the presence of E. histolytica was further studied by Cornick and colleagues who identified vesicle-associated membrane protein 8 (VAMP8) present on mucin granules as orchestrating regulated exocytosis in human goblet cells in response to the presence of E. histolytica (Cornick et al. 2017). In Vamp8−/− mice, E. histolytica induced enhanced killing of epithelial cells and aggressive proinflammatory response with elevated levels of IL-1α, IL-1β and TNF-α secretion, highlighting the downstream consequences of improper mucin secretion in mucosal barrier defence. Taken together, these results demonstrate the critical involvement of Muc2 in host protection from nematode infection, by constituting an effective physical and biological barrier against pathogenic infection. Muc1−/− mouse model Mice impaired in the production of cell surface mucins have also been engineered. The Muc1−/− mouse model revealed the role played by Muc1 in H. pylori infection, a pathogen involved in gastric ulcers and adenocarcinoma (McGuckin et al. 2007; Linden et al. 2009). Muc1−/− mice displayed a 5-fold increase in H. pylori colonisation as compared to wild type mice (McGuckin et al. 2007). This study further demonstrated the ability of H. pylori to bind to purified Muc1 in vitro, suggesting that Muc1 limits the access of H. pylori to the epithelial surface thereby providing protection from infection and proinflammatory bacterial products. Muc1 deficiency also resulted in increased epithelial cell apoptosis in H. pylori infected mice (Linden et al. 2009). More recently, the long-term consequence of Muc1 deficiency on H. pylori pathogenesis was investigated in Muc1−/− mice (Ng et al. 2016). Muc1−/− mice began to die 6 months after H. pylori challenge, indicating that a deficiency in Muc1 leads to lethal infection. This study also revealed that Muc1 was an important, previously unidentified negative regulator of the NLRP3 inflammasome, and loss of this regulation resulted in the development of severe pathology (Ng et al. 2016). Consistent with these studies, Muc1−/− mice have a higher rate of systemic infection in a murine C. jejuni model of gastroenteritis (McAuley et al. 2007). Muc13−/− mouse model The MUC13 transmembrane mucin is highly and constitutively expressed in the small and large intestines and MUC13 polymorphisms have been associated with human IBD and susceptibility to E. coli infection in pigs. While Muc13-deficient mice did not show intestinal pathology, they developed more severe acute colitis than wild type mice after DSS challenge, as reflected by increased weight loss, rectal bleeding, diarrhoea and histological colitis scores (Sheng et al. 2011). Mouse models with an altered mucin glycosylation Loss of O-glycans impairs the expression and function of several intestinal mucins, thereafter causing more profound defects in the function of the intestinal barrier than a flaw caused by the deficiency of an individual mucin. A number of transgenic mouse models have been developed to decipher the mechanisms underpinning the role of mucin glycosylation in gut homeostasis. Mice lacking core 3–derived O-glycans (also known as C3GnT−/− mice) display a substantial reduction of Muc2 protein and an increased susceptibility to DSS-induced colitis and accelerated colorectal tumorigenesis (An et al. 2007). In addition, core 3 O-glycosylation was shown to play a major role in controlling Salmonella intestinal burdens in C3GnT−/− mice (Zarepour et al. 2013). Similarly, C2GnT2−/− mice (mice lacking core 2-derived O-glycans) displayed an increased susceptibility to DSS-induced colitis but with no change in Muc2 expression (Stone et al. 2009). Mice with intestinal epithelial cell–specific deficiency of core 1–derived O-glycans (IEC C1galt1−/−) develop spontaneous colitis (Fu et al. 2011). Mice lacking both core 1- and core 3-derived O-glycans (DKO mice) have an impaired mucus barrier function and develop colitis-associated colon cancer in which the dysbiotic microbiota promote inflammation and cancer (Bergstrom et al. 2016). In a water avoidance model in rats, psychological stress lead to less-protective mucus layer. In particular, O-glycosylation of mucins was strongly affected and these changes were associated with flattening and loss of the mucus layer cohesive properties (Da Silva et al. 2014). Altogether, these data suggest that the lack of a proper O-glycosylation impairs Muc2 expression or secretion and alters gut barrier function of the mucus layer. In addition to modifications of mucin core glycans, mouse models have been developed targeting epitope modifications of the mucin glycans chains. Dawson and colleagues reported that deletion of the sulfate transporter NaS1 in mice (Nas1−/− mice) resulting in a decrease in mucin sulfation, enhanced susceptibility to experimental DSS colitis and systemic infection by C. jejuni (Dawson et al. 2009). In addition, mice with a deletion of the sulfo-transferase GlcNAc6ST2 enzyme adding sulfate to GlcNAC residues on O-mucin glycan chains exhibited an increased susceptibility to DSS-induced colitis (Tobisawa et al. 2010). Mice deleted for Sat-1 (sulphate anion transporter-1) were more susceptible to chronic infection by parasite T. muris (Hasnain et al. 2017). Collectively, these findings indicate that mucin abnormalities can initiate the onset of inflammatory related diseases in the gut. In addition to mice harbouring a deletion in genes encoding proteins directly involved in mucin expression or glycosylation, several transgenic mouse models have been shown to display alterations in mucus properties. These include the Winnie and Eeyore mice which carry single missense mutations in two different D-domains of Muc2 (Heazlewood et al. 2008). These mice display fewer goblet cells and a reduction in secreted mucus with O-glycosylated and non-O-glycosylated Muc2. The misfolding results in endoplasmic reticulum stress, goblet cell apoptosis, depletion of the secreted mucus layer and development of chronic intestinal inflammation (Heazlewood et al. 2008). In combination with mucus, a large population of intraepithelial lymphocytes (IELs) bearing the γδ T cell receptor is mediating immune protection against invading bacteria. In γδ T-cell-deficient (TCRδ−/−) mice, mucin expression and glycosylation is altered, mucus-secreting goblet cells are significantly reduced in number and those animals are more prone to DSS-induced colitis (Kober et al. 2014). Mouse models deficient in TLR5, IL-10 and Sodium hydrogen antiporter 3 (Slc9a3 or Nhe3) revealed bacteria in contact with the epithelium. Additional analysis of the less inflamed IL-10−/− mice revealed a thicker mucus layer but a more penetrable inner mucus allowing bacteria to penetrate and reach the epithelium (Johansson et al. 2014). Non-rodent models Differences in mucus thickness and composition have been observed between rats, pigs and rabbits, and suggests that the pig mucus pattern resembles more closely that of humans (Varum et al. 2012). The zebrafish larva is an emerging model system for investigating components of the innate immune system, including mucus physiology. It has been shown that five gel-forming secreted mucin genes are found in zebrafish with a high degree of homology to other vertebrate mucins regarding their genomic and protein domain organisation, as well as their tissue specific expression (Jevtov et al. 2014). Limitations of current experimental models involving mucus and future challenges Given the importance of the gut microbiota as a modulator of health and disease, increasing attention has been devoted to the role played by mucus in the interaction with gut bacteria (Juge 2019). As described above (section 4), various experimental models increasing in complexity from simple in vitro assays to cell lines, organ-on-chips, in vitro colon fermentation systems or animal models have been developed and successfully applied to the study of gut microbe-mucus interactions. However, one of the limitations common to most in vitro models is the origin of mucins used to assess the interactions with bacteria doubled by the inability to reproduce a colonic mucus gel recapitulating the in vivo situation. This is important as the nature and origin of purified mucins used in these assays greatly influence the outcome of binding as demonstrated using microtitre plate (Owen et al. 2017), dot-blot (Ringot-Destrez et al. 2018) or mucin microarrays (Clyne et al. 2017). Mucin glycosylation plays a critical role in the interaction between gut bacteria and mucus and significant glycosylation differences occur between purified mucins used in in vitro assays from different sites of the murine GI tract or from goblet cells (e.g. LS174T), as analysed by mass spectrometry (Leclaire et al. 2018; Ringot-Destrez et al. 2018). In addition, the purification steps alter the properties of native glycoproteins and purified mucins used in these assays lack the ability to form viscoelastic hydrogels (Kocevar-Nared, Kristl and Smid-Korbar 1997). A similar situation occurs with mucus secreting cell lines where the type of mucins and structure of mucus differ from the colonic environment. For example, the HT29 cell line secretes mostly MUC5AC whereas MUC2 is the main mucin secreted in the small and large intestines. These differences are due to the use of cancer cells which show an alteration in the expression and glycosylation of mucins. In addition, the production of mucus by epithelial cell lines can be influenced by culture conditions. For example, growing cells on Transwell filters with a small amount of apical medium (semi-wet interface culture) in combination with mechanical stimulation (on a rocking platform) and addition of the Notch γ-secretase inhibitor DAPT resulted in polarisation and secretion of MUC2 and MUC5AC by HT29 MTX-P8, HT29 MTX-E12 and LS513 cells (Navabi, McGuckin and Lindén 2013). Additionally, the mucus produced by goblet cells in in vitro co-culture cell models is not continuous nor homogenate which is not fully representative of the in vivo situation. Lastly, the formation of the bi-layered mucus found in the colon remains a challenge in these models. Novel strategies such as multiple cell layers, 2D-organoid techniques or Organ-on-a-Chip devices are currently being developed to better mimic the human intestinal epithelial microenvironment. Such multiple cell models exhibit intestinal villus morphogenesis associated with mucus production. These models are also needed to recapitulate antimicrobial defense and inflammatory reactions normally occurring in mucosal tissues. Another advantage of these systems is that, unlike cell lines, organoids can be used to evaluate long-term interactions between mucus and gut microbes. However, these more advanced biopsy- based models remain low throughput and expensive as compared to in vitro assays and are limited by the availability and variability of clinical specimens. In addition, to the host side, several microbial factors must be taken into consideration when assaying the interactions between the gut bacteria and mucus. These include the handling and labelling microbial cells which may affect the surface molecular determinants potentially involved in mucus/mucin interactions (e.g. cell-surface adhesins, pili or flagella) (Chagnot et al. 2014). The growth conditions (e.g. growth media with different nutrient compositions; temperature, pH, osmolarity or redox potential) can also influence the expression of the bacterial receptors mediating the interactions with mucins. To date, most studies have focused on the interactions between mucus and probiotic or pathogenic strains and assessing strictly anaerobe gut symbionts or complex microbial communities remain a challenge in this field of research. An alternative to the systems described above is the use of dynamic in vitro fermentation models of the human gut, such as the M-SHIME (Marzorati et al. 2014) or DP-M-SHIME (De Paepe et al. 2018) models. In these in vitro colonic models, the introduction of mucin-covered beads allows to study the long-term in vitro microbial colonisation of mucin, in the presence of a complex anaerobic intestinal microbiota (Marzorati et al. 2014; Shah et al. 2016). These models provide a mean to study gut microbiota functionality and niche differentiation, during treatments with xenobiotics (for example antibiotics, synthetic chemicals such as food additives, environmental pollutants like persistent organic pollutants (POPs)), pathogens or functional foods. Future developments in this field will be the introduction of mucus secretion and/or a mucus surface layer in dynamic in vitro models of the upper GI tract, such as in the gastric and small intestinal TNO Gastro-Intestinal model (TIM) (Guerra et al. 2012). This is important so to take into account the successive stressful events (e.g. acidic gastric pH, bile salts) that commensal or pathogenic microbes undergo in the human GI tract before reaching the intestinal epithelium and that may greatly influence their physiological stage, virulence and/or activity. However, as mentioned above, a limitation of these in vitro GI models is that they rely on commercially available mucins used for the mucin bead technology. These secretory mucins, usually MUC5AC and MUC6 porcine gut gastric mucin, differ in terms of structure and glycosylation from intestinal MUC2 and cannot form a bi-layered mucus gel. It has been proposed that in the future, in vitro engineered mucus may be used to mimic human-derived mucus in a more reproducible manner. The colonic in vitro models could also be improved by including immunoglobulins, specific antimicrobial peptides, or secreted phosphatidylcholine, which have been shown to modulate mucus surface properties, thereby influencing bacterial adhesion (Martens, Neumann and Desai 2018). Future in vitro colon models should also better mimic the in vivo transit, and particularly retrograde movements (Hiroz et al. 2009), as back-flow was recently suggested to be crucial for the persistence of gut microbes in the GI tract (Cremer et al. 2016). Current technological advances include the coupling of these fermentation models to intestinal epithelial cells or more complex units such as the HMI module. A next step will be to couple the digestive/fermentation models with enteroids/colonoids or HIO. However, despite their increased complexity, most of these approaches remain limited by the absence of important host functions, such as variable peristalsis-like motions. This is a critical limitation because mechanical deformations resulting from peristalsis both influence normal epithelial cell differentiation and control microbial overgrowth in the living intestine (Gayer and Basson 2009; Benam et al. 2015). The development of microfluidic systems and organ-on-chips is currently addressing this important technological gap (Kim and Ingber 2013; Kim et al. 2016). The development of these advanced in vitro systems is essential to help reduce dependence on animal studies. Due to the invasive nature of the experiments, the mechanisms underpinning microbe-mucus interactions in vivo have mainly been investigated in animal models, mostly rodents. Genetically engineered mice impaired in mucin secretion or glycosylation have been instrumental to decipher the role of mucins and mucus in the protection of the intestinal epithelium and the interactions between pathogenic bacteria, commensal microbiota and the mucus barrier. However, although the domain organisation and expression pattern of mucins appear largely conserved between human and mouse (Joshi et al. 2015), mucin glycosylation and gut microbiota (Nguyen et al. 2015) differ between these two species. It has been speculated that differences in mucin glycosylation between mammalian species may underlie some of the differences in infectivity and/or pathogenicity for individual microbial pathogens (Linden et al. 2008) or the different commensal microbiota (Thomsson et al. 2012). Therefore, caution should be applied when translating data obtained in mouse models to humans. Lastly, unlike in vitro assays, in vivo studies are restricted to end-point measurements. Recent years have witnessed unprecedented technological advances in the development of in vitro GI models that more closely resemble the gut mucosal interface. Our next challenge will be to simulate these models at different stages of development or disease conditions (e.g. IBD, obesity or CF). Special attention should be paid to inter-individual differences and intra-individual variability in gut microbiota composition and intestinal biopsies from different donors or patients. This is important to better understand the role of gut microbe–mucus interactions in the aetiology of a particular disease or condition and determine the microbial and biochemical signature that could differentiate between diseased and healthy status. In particular, more research is warranted to determine how the physicochemical properties and/or thickness of the mucus layer and mucin glycosylation are altered during a specific disease. 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TI - Experimental models to study intestinal microbes–mucus interactions in health and disease
JF - FEMS Microbiology Reviews
DO - 10.1093/femsre/fuz013
DA - 2019-09-01
UR - https://www.deepdyve.com/lp/oxford-university-press/experimental-models-to-study-intestinal-microbes-mucus-interactions-in-1kXhckioPz
SP - 457
VL - 43
IS - 5
DP - DeepDyve
ER -