The immune interactions of gut glycans and microbiota in health and disease

Abstract

The human digestive system harbors a vast diversity of commensal bacteria and maintains a symbiotic relationship with them. However, imbalances in the gut microbiota accompany various diseases, such as inflammatory bowel diseases (IBDs) and colorectal cancers (CRCs), which significantly impact the well-being of populations globally. Glycosylation of the mucus layer is a crucial factor that plays a critical role in maintaining the homeostatic environment in the gut. This review delves into how the gut microbiota, immune cells, and gut mucus layer work together to establish a balanced gut environment. Specifically, the role of glycosylation in regulating immune cell responses and mucus metabolism in this process is examined.

1 |. INTRODUCTION

The gut microbiota encompasses many beneficial bacteria in the human gastrointestinal (GI) system. These symbiotic microorganisms play a crucial role in maintaining the overall health and wellness of the human body, aiding in digestion, nutrient absorption, and immune system regulation (Fan & Pedersen, 2021). The study of gut microbiota has increasingly gained attention, particularly in the last two decades. Over time, the terms microbiota and microbiome have been used interchangeably. In this article, we use microbiota to refer to bacterial taxa and microbiome to refer to the microbial metagenome (Ursell et al., 2012).

The human gut microbiota has a complex network of diverse microbes, including bacteria, fungi, protozoa, archaea, and viruses (Erturk-Hasdemir & Kasper, 2013; Mafra et al., 2022; Wrede et al., 2012). Through mutualism, the gut microbiota plays central roles in protection against enteric pathogens, maturation and homeostasis of the immune system, regulation of the immune response, energy metabolism, and production of essential nutrients (Candela et al., 2008; Fukuda et al., 2011; Round & Mazmanian, 2009; Sonnenburg et al., 2005). Specific changes in the gut microbiota can be associated with numerous conditions, including diseases such as ulcerative colitis (UC), Crohn’s disease (CD), and colorectal cancer (CRC) (Table 1) (Chung & Kasper, 2010; Duan & Kasper, 2011; Garrett, 2019; Khoruts et al., 2010; Ursell et al., 2012).

TABLE 1.

Select bacterial taxa and mucosal alterations in UC, CD, and CRC.

	Bacterial taxa		Mucosal alterations
Disease	Increased relative abundance	Decreased relative abundance	Increased	Decreased
Ulcerative Colitis (UC)	Bacteroides fragilis (Vich Vila et al., 2018) Clostridium hathewayi (Lloyd-Price et al., 2019) Clostridium bolteae (Lloyd-Price et al., 2019) Escherichia coli (Pittayanon et al., 2020)	Akkermansia muciniphila (Pittayanon et al., 2020) Bifidobacterium longum (Vich Vila et al., 2018) Eubacterium rectale (Vich Vila et al., 2018) Eubacterium rectum (Pittayanon et al., 2020) Faecalibacterium prausnitzii (Vich Vila et al., 2018)	MUC1, MUC16 (Bankole et al., 2021; Grondin et al., 2020) MUC5AC presence (Kang et al., 2022) Expression of truncated or shortened O-glycans (Kudelka et al., 2020)	MUC2, MUC9, MUC20 (Dorofeyev et al., 2013; Van der Sluis et al., 2006; Yamamoto-Furusho et al., 2012, 2015) Mucus thickness, sulfation, and sialylation (Kang et al., 2022) Goblet cell number (Gersemann et al., 2009, Kang et al., 2022)
Crohn’s Disease (CD)	Actinomyces (Pittayanon et al., 2020) Bacteroides fragilis (Vich Vila et al., 2018) Clostridium hathewayi (Lloyd-Price et al., 2019) Clostridium bolteae (Lloyd-Price et al., 2019) Escherichia coli (Pittayanon et al., 2020)	Clostridium leptum (Pittayanon et al., 2020) Eubacterium rectale (Vich Vila et al., 2018) Faecalibacterium prausnitzii (Vich Vila et al., 2018)	MUC1 (in recurrent cases) (Hashash et al., 2021) MUC5AC presence (Kang et al., 2022) Mucus thickness (or no change) (Kang et al., 2022) Non-sense mutation of FUT2 (Battat et al., 2022)	MUC2, MUC3, MUC4, MUC5B, MUC7 (Grondin et al., 2020; Hashash et al., 2021) Goblet cell number (Gersemann et al., 2009, Kang et al., 2022)
Colorectal Cancer (CRC)	Bacteroides fragilis (Vich Vila et al., 2018) Enterococcus faecalis (Wang et al., 2012) Escherichia coli (Veziant et al., 2016) Fusobacterium nucleatum (Mima et al., 2016) Streptococcus bovis (Gupta et al., 2010) Streptococcus gallolyticus (Kumar et al., 2017) Solobacterium moorei (Wong & Yu, 2023) Parvominas micra (Zhao et al., 2022) Peptostreptococcus anaerobius (Wong & Yu, 2023)	Alistipes (Wang et al., 2012) Eubacterium (Wang et al., 2012) Parasutterella (Wang et al., 2012) Roseburia (Wang et al., 2012)	MUC1 (Li et al., 2019) MUC5AC (APC pathway) (Rico et al., 2021)	MUC2 (Cecchini et al., 2019) MUC5AC (BRAF pathway) (Pothuraju et al., 2020) MUC4 (Pothuraju et al., 2020) MUC17 (in BRAF pathway) (Pothuraju et al., 2020)

Glycans and glycoconjugates (i.e., glycoproteins, glycolipids, proteoglycans, etc.) are found in all living organisms and displayed in diverse and distinct structures, combinations, and sizes (Kudelka et al., 2015). They play essential roles in cell signaling, energy metabolism, and structural support (Varki, 2017). Within the GI tract, glycans are primarily found on mucin glycoproteins and dietary fibers, metabolized by the gut microbiota (Koropatkin et al., 2012; Slavin, 2013; Varki, 2017). Select species such as Bacteroides thetaiotaomicron are known to degrade both mucin glycans and dietary fibers (Koropatkin et al., 2012). Therefore, an in-depth understanding of the relationship between gut microbiota and dietary glycans may help identify pathophysiologic mechanisms of diseases associated with dysbiosis of gut microbiota.

This review discusses the complexity and function of gut microbiota from a bacterial perspective. We explore how various elements like the gut microbiota, submucosal immune system, and gut mucus layer work together to regulate gut immunity to establish and maintain a homeostatic environment. We also examine the significance of glycan and mucus metabolism in this process.

2 |. THE GUT MICROBIOTA

The human gut microbiota (hGM), also known as the “forgotten organ,” is a highly diverse composition of microbes that increase in density and diversity from proximal to the distal gut (Fan & Pedersen, 2021; O’Hara & Shanahan, 2006; Sekirov et al., 2010). The GI tract is colonized by trillions of bacteria, with over 1000 different bacterial species (Thursby & Juge, 2017). These commensal bacteria play a crucial role in maintaining the homeostatic environment in the gut (Zhang et al., 2022).

Newborns have a functionally and structurally immature GI system (Wagner et al., 2008). Beginning at birth, the GI tract is inoculated with microorganisms, and the mode of delivery affects the microbial composition (Ajslev et al., 2011; Collado et al., 2012; Dominguez-Bello et al., 2011). The gut microbiota diversifies early on and resembles that of an adult by age one (Palmer et al., 2007). The mature, healthy gut microbiota is primarily composed of phyla such as Bacteroidetes, Firmicutes, Proteobacteria, and Actinobacteria, Bacteroidetes and Firmicutes being the most dominant phyla (Arumugam et al., 2011; Eckburg et al., 2005; Rinninella et al., 2019). These symbiotic bacteria have various roles in metabolism, such as producing essential vitamins, maintaining tissue homeostasis, protecting the host against opportunistic pathogens, and producing short-chain fatty acids (SCFAs) from the indigestible carbohydrates and the mucins to be used by the host as an energy source (Cummings, 1983; Smith et al., 2007; Sommer & Bäckhed, 2013). The effects of microbiota greatly depend on its composition, which varies due to factors such as age, diet, and antibiotic usage (Biagi et al., 2010; Dethlefsen et al., 2008; Dethlefsen & Relman, 2011; Jernberg et al., 2010; Willing et al., 2011; Yatsunenko et al., 2012).

Changes in the composition of the gut microbiota are associated with diseases such as IBDs, CRCs, and metabolic syndrome as a contributing factor in the development of these multifactorial diseases (Belkaid & Hand, 2014; Le Chatelier et al., 2013; Wong & Yu, 2023). The microbiota’s composition also changes with the host’s physiological state. For example, obese individuals harbor less diverse microbiota than lean individuals (Ley et al., 2006; Turnbaugh et al., 2006). Improved overall hygiene due to urbanization may decrease the diversity of bacterial genera in the gut, especially Bacteroides, Prevotella, and Lactobacilli, as Fan and Pedersen reviewed (Fan & Pedersen, 2021). This decreased bacterial diversity and bacterial gene complexity can be linked to insulin resistance, inflammation, and eventually dyslipidemia (Le Chatelier et al., 2013). Antibiotics, while essential in medical practice, can cause GI side effects, such as hypersensitivity and antibiotic-associated diarrhea (Wiström et al., 2001). A short course of broad-spectrum antibiotic treatment can lead to dysbiosis and a decrease in some commensal bacteria in adults (Palleja et al., 2018).

Patients with IBDs, such as CD, UC, and indeterminate colitis, typically have low microbial diversity in their gut microbiota (Table 1) (Kostic et al., 2014). Irritable bowel syndrome (IBS), Clostridioides difficile (C. difficile)-associated colitis, and acute diarrhea are also associated with an alteration in fecal microbiota composition (Carroll et al., 2011; Chang et al., 2008; Young & Schmidt, 2004). Dysbiosis is a well-established phenomenon in CRCs, the second leading cause of cancer-related deaths in the United States, as demonstrated by large-scale human studies (Feng et al., 2015; Siegel et al., 2022). Increased numbers of resident bacteria such as Bacteroides fragilis (B. fragilis), Escherichia coli (E. coli), and Streptococcus gallolyticus (S. gallolyticus) were observed in CRC patients (Andres-Franch et al., 2017; Arthur et al., 2012; Wong & Yu, 2023; Wu et al., 2009). In addition, specific pathogenic bacterial populations are found to be enriched in CRC patients, most notably Fusobacterium nucleatum (F. nucleatum), Solobacterium moorei (S. moorei), Peptostreptococcus anaerobius (P. anaerobius), and Parvimonas micra (P. micra) (Table 1) (Wong & Yu, 2023). Interestingly, in patients with colorectal adenomas, there is an increased number of pathogens like F. nucleatum and S. moorei (Yachida et al., 2019).

The epithelial and the immune cells of the GI tract mediate mucosal immune responses to regulate the gut microbiota (Goto & Ivanov, 2013). The immune responses include mucus composition, IgA, and antimicrobial secretion such as RegIIIγ (an antibacterial lectin produced by enterocytes and Paneth cells) and defensins (Bergstrom & Xia, 2013; Peterson et al., 2007; Salzman et al., 2010; Vaishnava et al., 2011; Wang et al., 2021).

3 |. GUT IMMUNITY IS REGULATED BY THE GUT MICROBIOTA, GLYCANS, AND THE IMMUNE SYSTEM

The GI system has a complex and multifaceted immune regulation. The gut-associated lymphoid tissues (GALTs) develop before birth, including mesenteric lymph nodes (MLNs), appendix, Peyer’s patches, and isolated lymphoid follicles (Eberl & Lochner, 2009; Mörbe et al., 2021). The gut microbiota and the immune system work together to establish and maintain homeostasis (Figure 1a).

FIGURE 1.

Gut immunity is regulated through the interactions between the immune system and the gut microbiota. (a) Immune regulation of a healthy gut microenvironment. (1) IgA secreted by plasma cells regulates bacterial concentration in the mucosal layer. (2) MAMPs trigger TLR-Myd88 signaling to induce antimicrobial peptides (i.e., RegIIIγ and α-defensins), which limit bacterial access to mucosal and epithelial layers. (3) Mincle recognition of commensal bacteria in the gut inhibits IL-17 cytokine and Th17 cell responses through IL-6 and IL-23p19 cytokines. (4) Anti-inflammatory responses maintain the homeostatic gut environment through Treg cell activity, Siglec activity, tolerogenic dendritic cells and macrophages, and galectin-1 activation. (b) Immune regulation of an unhealthy gut microenvironment. (1) CX3CR1hi mononuclear phagocytes transport commensal bacteria to CD103+ dendritic cells in the mesenteric lymph nodes, inducing pro-inflammatory T-cell responses and increased IgA production and aggregation. (2) Downregulated Mincle levels and Mincle-dependent immune evasion by opportunistic pathobionts. (3) DC-SIGN-dependent immune evasion enabled by Th1 pro-inflammatory response inhibition after recognition of pathogenic/pathobiont bacteria. (4) Additional pro-inflammatory responses induce the inflammatory gut environment by Th17 cell activity, Siglec activity, immunogenic dendritic cells and macrophages, and galectin-3, -4, and -9 activity.

3.1 |. Immune regulation via the gut microbiota and lymphatic cells

The gut microbiota plays an essential role in the immune system maturation and homeostasis since GALT maturation, T-cell activation, and plasma cell recruitment depend on the microbiota-derived signals (Eberl & Lochner, 2009; Olszak et al., 2012; Sommer & Bäckhed, 2013). Immune cells in the intestine can be associated with two different functional sites: inductive sites (Peyer’s patches, MLNs, and lymphoid follicles) and effector sites (lamina propria and epithelia) (Garrett et al., 2010). Studies have shown that germ-free mice with immature mucus layers are more susceptible to GI infection with pathogenic bacteria due to their smaller MLNs and lamina propria, reduced T-helper 17 (Th17) cells, and IgA (Hapfelmeier et al., 2010; Hooper et al., 2001; Ivanov et al., 2009; Smith et al., 2007). However, these abnormalities can be ameliorated with conventionalization of the gut with a complex murine microbiota (Erturk-Hasdemir & Kasper, 2013). While the commensal bacteria boost the host’s digestive system efficiency, colonization with pathogens can lead to inflammation and sepsis (Hooper et al., 2012). Alterations in the hGM can contribute to the development of IBDs (Figure 1b) (Shanahan, 2012). The intricate interplay between the human immune system and the microbiota has led to the evolution of metabolic benefits for both (McFall-Ngai, 2007). Pathogenic and non-pathogenic organisms are both sensed by the pattern recognition receptors (PRRs), which include Toll-like receptors (TLRs), C-type lectin receptors (CLRs), and Nod-like receptors (NLRs) (Kawai & Akira, 2011). PRR activation results in a cascade of pro-inflammatory responses due to the recognition of microbe-associated molecular patterns (MAMPs) (Kawai & Akira, 2011). Pili, flagella, and peptidoglycans are examples of known MAMPs. Some commensals, for instance, Lachnospiraceae, produce silent flagellins that are weak stimulants of TLR5 to evade the immune response; alternatively, the same receptor recognizes the Salmonella flagellin to generate an immune response (Clasen et al., 2023).

The phagocytes residing in the lamina propria, such as macrophages and dendritic cells (DCs), are hyporesponsive to TLR ligands from commensal bacteria, preventing the production of immune responses like Tumor Necrosis Factor (TNF) or Interleukin (IL)-6 (Erturk-Hasdemir & Kasper, 2013; Franchi et al., 2012; Smythies et al., 2005). However, these same innate immune cells produce pro-inflammatory cytokines such as pro-IL-1-β when exposed to pathogenic bacteria such as Salmonella enterica serovar Typhimurium (S. Tm) (Franchi et al., 2012). This, in turn, induces the production of IL1-β through the NLRC4 inflammasome via the presence of the functional type III secretion system, which does not rely on TLR signaling (Figure 1) (Franchi et al., 2012). Intercrypt macrophages in lamina propria sense S. Tm lipopolysaccharides (LPS) via TLR4, and secrete TNF to induce epithelial NF-κB signaling, elucidating the role of inflammasomes and the organization of TLR4 responses in the murine gut mucosa (Hausmann et al., 2021). In addition, the NLRC4 defense relies mainly on gut-epithelial cells, while the lamina propria phagocytes act as the main switches for LPS-TLR4 signaling (Fattinger et al., 2021). This mechanism plays an important role in restricting the intraepithelial pathogen proliferation (Sellin et al., 2014). In a dysbiotic environment, non-invasive bacteria are trafficked to the CD103+ DCs in the mesenteric lymph nodes by CX3CR1^hi mononuclear phagocytes in a CCR7-dependent manner. This results in T-cell activation and increased IgA production due to a lack of commensal bacteria-induced Myd88 activation (Figure 1b) (Diehl et al., 2013; Hapfelmeier et al., 2008; Mazzini et al., 2014).

The commensal microbiota promotes the development of regulatory T cells that play an essential role in immune tolerance (Belkaid & Hand, 2014; Round & Mazmanian, 2009). Within the GI tract, antigen-presenting cells (APCs) process the bacterial antigens and then present them to aid in the transformation of naive CD4⁺ T cell to a Th2 cell (Figure 1a) (Gurram & Zhu, 2019). This process allows Th2 cells to secrete effector cytokines like IL-13 (Gurram & Zhu, 2019). Germ-free mice were shown to have a CD4⁺ T-cell imbalance with a Th2 bias (Mazmanian et al., 2005), and mono-colonization of these mice with the commensal bacterium B. fragilis can reestablish the Th1 and Th2 balance (Mazmanian et al., 2005). Finally, Paneth cells in the small intestine secrete α-defensins, a class of antimicrobial peptides, which are the pre-dominant antibacterial factors against enteric pathogenic bacteria (Figure 1a) (Mazmanian et al., 2005).

3.2 |. Immune regulation via the epithelial barrier

The gut epithelial layer is a physical and immune barrier essential to immune regulation. It contains goblet cells (produce mucus), M cells (present in Peyer’s patches and lymphoid follicles, sensing and transporting microbes), enterocytes (absorption), stromal cells (tissue regeneration and wound repair of epithelium), and Paneth cells (secrete antimicrobial molecules like lysozyme, store and release zinc, and sense microbial products via Myd88-dependent pathways) (Johansson et al., 2008; Kyd & Cripps, 2008; Stappenbeck & Miyoshi, 2009; Vaishnava et al., 2008). Commensal bacteria cause an auto-activation of Myd88, an adaptor protein that plays a central role in TLR activation, which limits bacterial access to the mucosal layer (Figure 1a) (Deguine & Barton, 2014; Vaishnava et al., 2008). The epithelial tight junctions also contribute to immune regulation in the gut (Beau et al., 2007; Nusrat et al., 2001; Sakaguchi et al., 2002). Pathogens like Rotaviruses, C. difficile, Shigella flexneri, and Salmonella typhimurium may cause diseases by disrupting tight junctions through altering the distribution of occludin, altering the tight junction microdomain localization, targeting claudins, and altering the localization of tight and adherent junction proteins (Figure 2b) (Beau et al., 2007; Nusrat et al., 2001; Sakaguchi et al., 2002; Tafazoli et al., 2003).

FIGURE 2.

The gut microbiota interacting with mucosal-epithelial barrier. (a) Intact mucus layer, glycocalyx, and epithelial tight junctions restrict bacterial access to the epithelial barrier. Mucin glycans aid in colonization by acting as attachment points and as sources of nutrients. Mucin glycan sialylation protects the integrity of the mucus layer from bacterial proteolytic degradation. Fucosylation of mucin through FUT2 promotes the colonization of commensal bacteria and reduces opportunistic bacteria colonization. (b) Impaired/degraded mucus layer, glycocalyx, and epithelial barrier grant access to commensal and pathogenic/pathobiont bacteria to permeate beyond the barrier. A decreased amount of mucin-producing goblet cells and altered mucin glycosylation and sialylation lead to an impaired mucus layer and glycocalyx. (c) Commensal bacteria digest dietary (i.e., fiber) and host glycans through CAZymes to generate monosaccharides, disaccharides, and SCFAs, which are subsequently used as a nutrient and energy resource by the microbiota and host. (d) Altered digestion of dietary and host glycans leads to altered production of monosaccharides, disaccharides, and SCFA.

3.3 |. Immune regulation via IgA

IgA can be found in two forms: mucosal IgA and serum IgA. Mucosal IgA has a polymeric structure and is concentrated in the outer layer of mucus (Rogier et al., 2014). Mucosal IgA is produced by plasma cells within the germinal centers of the Peyer’s patches and binds to microbial antigens with a very high affinity (Pabst, 2012; Woof & Russell, 2011). Secretory IgA, cleaved from the mucosal polymeric IgA, interacts with many antigens in the lumen of the intestine (Pabst & Izcue, 2022). IgA has numerous roles in gut homeostasis, which include entrapping antigens in the mucus, reducing invasive properties of bacteria, excreting antigens from the lamina propria into the intestinal lumen, and reducing bacterial motility (Pabst, 2012). IgA’s pro- and anti-inflammatory effects are mediated by its binding to the FcαRI. While the FcαRI binding of soluble IgA mediates the anti-inflammatory responses, binding of the aggregated form mediates the pro-inflammatory responses (Ding et al., 2022). IgA helps maintain gut immune regulation in a non-specific fashion via a process called immune exclusion (Corthésy, 2013). This process depends on IgA’s ability to prevent microbial access to the epithelial layer with a chain of events called agglutination (bacterial clump formation), entrapment, and clearance (Figure 1) (Mantis et al., 2011).

Agglutination is effective at high microbial densities (≥10⁸) in the gut lumen (Moor et al., 2017). However, typical infections present with lower densities (10⁰–10⁷) of dividing bacteria. High-avidity IgA protects the host by a mechanism called IgA-mediated cross-linking enchainment of growing bacteria (Moor et al., 2017). Additionally, the zymogen granule protein 16 (ZG16) in the mucus layer binds to peptidoglycan structures, such as on Lactobacillus jensenii, and aggregates these bacteria, another mechanism for how the mucus layer keeps microbes away from the colonic epithelial layer (Bergström et al., 2016).

3.4 |. Immune regulation via RegIII

The Reg gene family encodes a diverse group of secreted proteins, further classified into subgroups (I, II, III, IV) containing conserved sequence motifs found in C-type lectin carbohydrate recognition domains (CRDs) (Cash et al., 2006). Increased expression of RegIII depends on factors such as surgery, nutrition, and inflammation due to bacterial invasion or mucosal damage (Cash et al., 2006; Everard et al., 2014; Zhao et al., 2018). RegIIIγ (the mouse homolog of human REG3α) expression also depends on the microbiota. A study demonstrated a significant decrease in RegIIIγ expression in germ-free mice compared to wild-type mice (Cash et al., 2006).

The C-type lectins of the RegIII family, secreted by enterocytes and Paneth cells primarily in the distal ileum, show bactericidal properties by restricting mucosal access of gram-positive bacteria to the small intestinal epithelium and by enabling spatial segregation (Paschall et al., 2023; Vaishnava et al., 2011). This protection is achieved through TLRs, which detect the microorganisms and activate Myd88 signaling (Figure 1a) (Vaishnava et al., 2011). Mice lacking Myd88 showed a loss of ~50 μm zone of inhibition over the ileal epithelium and an increase in mucosa-associated bacterial numbers in the small intestine per FISH analysis and qPCR determination of 16 s rRNA gene copy numbers (Vaishnava et al., 2011). Additionally, in RegIIIγ knockout mice, the number of gram-positive bacteria increased in the small intestine compared to the wild-type littermates, while the gram-negative bacterial loads stayed the same (Vaishnava et al., 2011).

3.5 |. Immune regulation via lectins

Lectins on immune cell surfaces can detect bacterial glycans as a primary class of PRRs. C-type lectins, Siglecs, and galectins are three major lectin families (Prado Acosta & Lepenies, 2019).

In Peyer’s patches, macrophage-inducible C-type lectin (Mincle) recognizes commensal bacteria in the mucosa (Martínez-López et al., 2019). This recognition induces expression of IL-6 and IL-23p19 and thereby regulates Th17 differentiation and IL-17 secretion (Figure 1a) (Martínez-López et al., 2019). The same study demonstrated that Mincle-deficient mice develop systemic translocation of the gut microbiota, for instance, Proteobacteria to the liver from the gut (Martínez-López et al., 2019). Helicobacter pylori (H. pylori), a pathogen that colonizes gastric mucosa, was shown to interact with Mincle through its Lewis antigens of LPS and cause an anti-inflammatory response to reside in the host (Devi et al., 2015). H. pylori can also bind to DC-SIGN, a CLR, to evade immune responses by blocking the maturation of naive T-cells to Th1 cells (Figure 1b) (Gringhuis et al., 2009).

Sialic acid-binding immunoglobulin-like lectins (Siglecs) are membrane-bound receptors expressed on nearly all immune cells, and binding of bacterial products to these receptors may create both pro- and anti-inflammatory responses depending on the activated Siglec receptor type (Figure 1) (Kang et al., 2020). For instance, Neisseria gonorrhoeae recognition by Siglec-11 and Siglec-16 can lead to anti- and pro-inflammatory responses, respectively, while Siglec-10 recognition of Campylobacter jejuni (C. jejuni) flagella can promote an anti-inflammatory response (Landig et al., 2019; Prado Acosta & Lepenies, 2019; Stephenson et al., 2014).

Galectins, a family of secreted lectins with an affinity for beta-galactosides, are expressed by immune cells, including natural killers (NKs), DCs, macrophages, and activated T and B cells (Rabinovich et al., 2007; Vasta, 2009). Galectins can regulate adaptive immunity by influencing T-cell signaling and activation, and modulating immunosuppressive Treg function (Rabinovich & Toscano, 2009). During infections, galectin expression can vary, and interactions with bacteria and galectins can affect infection and sepsis (Ferreira et al., 2018; Lo et al., 2021). Galectin (Gal)-1 dampens Th1- and Th17-mediated responses, creating a Th2 dominant immune response (Stillman et al., 2006; Sundblad et al., 2018; Toscano et al., 2007). This effect is due to Th1 and Th17 cells expressing Gal-1 binding glycans; conversely, Th2 cells display α2,6-sialic acid-capped glycoproteins on their surfaces (Figure 1a) (Tsai et al., 2016). A study utilizing a 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis model in mice demonstrated that treating with recombinant Gal-1 improved the disease outcome (Tsai et al., 2016). Recombinant Gal-1 diminishes the effects of TNBS-related T cells (Santucci et al., 2003). Galectins can also recognize host glycans expressed on vacuoles that harbor intracellular pathogens and then induce autophagy (Li et al., 2020). A mouse study utilizing a Gal-3 knockout (KO) model demonstrated that when colitis was induced via dextran sulfate sodium administration, KO mice developed more severe colitis compared to wild-type littermates (Tsai et al., 2016). Treating the mice with Gal-3 ameliorated the effects of colitis (Tsai et al., 2016). A recent study demonstrated that Gal-4 in intraepithelial lymphocytes coats cytosolic Salmonella enterica serovar Worthington, inducing bacterial chain and aggregate formation (Li et al., 2023). This process restricts bacterial motility and helps potentiate the inflammasome activation (Li et al., 2023). Chemotherapy treatments using agents like Fludarabine and Busulfan can damage intestinal cells and increase T-cell activation and migration. Damaged organoids have been shown to possess increased Galectin-9, the key mediator for this chemotherapy-associated T-cell activity (Figure 1b) (Jansen et al., 2023).

4 |. ROLE OF GLYCANS IN MICROBIOTA HOMEOSTASIS

Glycans decorate all cell surfaces, such as mucin glycans and proteoglycans on host epithelial cells or capsular polysaccharides, lipopolysaccharides, or glycoproteins on bacterial cells (Hart & Copeland, 2010; van Kooyk & Rabinovich, 2008). The glycocalyx (combined coat of glycans on the host cell surfaces, acting as a protective barrier between the epithelial layer and mucus layer) is essential in mediating immune responses in the gut by separating epithelial cells from the microbiota (Figure 2a) (Ouwerkerk et al., 2013).

4.1 |. Intestinal glycans and mucus

4.1.1 |. Mucin structure and types

Glycans covalently attach to the polypeptide chains primarily through the amide nitrogen of asparagine side-chain (N-linked) and the hydroxyl group of serine/threonine side-chains (O-linked), and on rare occasions, the thiol group of cysteine side-chain (S-linked) (Maynard et al., 2016; Varki, 2017). In the colon, epithelial glycans are significant components of the intestinal mucus and regulators of the interaction between the gut microbiota and the gut epithelia (Arike et al., 2017). In the small intestine, glycocalyx primarily regulates these interactions (Layunta et al., 2021). The mucus comprises proteins, salts, lipids, immunological factors, and a hydrogel layer of mucins (MUCs) (Bansil & Turner, 2018). Mucins are glycoproteins that can be broadly classified as secreted or membrane-bound and are primarily produced by goblet cells or enterocytes, respectively (Dekker et al., 2002). They are heavily O-glycosylated (nearly 80%) by the host Golgi apparatus and contain large peptide domains with repeating proline, threonine, and serine amino acids (the PTS domain) (Figure 3) (Linden et al., 2008).¹⁷⁵ Mucin glycosylation and the number of repeats depend on the cell lineage, developmental stage, and anatomical section (Bergstrom et al., 2020; Larsson et al., 2011; Linden et al., 2008). These O-linked oligosaccharides are mainly composed of N-acetyl galactosamine (GalNAc), N-acetyl glucosamine (GlcNAc), galactose (Gal), sialic acid (Sia), and fucose (Fuc) (Cummings et al., 2022; Grondin et al., 2020; Linden et al., 2008). Increased glycosylation leads to increased water absorption and relative expansion of the mucus layer, which leads to a greater barrier (Grondin et al., 2020). In addition, O-glycosylation of mucins helps protect the mucus layer from bacterial proteases (Tran & Ten Hagen, 2013).

FIGURE 3.

MUC2 protein glycosylation in homeostatic (normal) gut environment versus unhealthy/dysbiotic gut environment. (a) MUC2 protein domain organization. The high density of O-glycosylation takes place in PTS domains. (b) AlphaFold depiction of MUC2 region from residues 1260 to 1460 (UniProt Primary Accession: Q02817). Protein structure is colored according to model confidence. Visualization of the predicted aligned error plot demonstrates high model confidence between residues 1300–1395, associated with the ordered domain highlighted in dark and light blue. Model confidence falters in regions associated with PTS-repeats, indicative of the inherent disordered state of the protein backbone and displayed in orange and yellow. MUC2 glycans observed in a homeostatic (normal) gut environment are densely packed. Glycan structures are elongated, sulfated, and fucosylated. MUC2 glycans observed in unhealthy/dysbiotic gut environment are sparse, short, altered, and aberrant with increased expression of T antigen, Tn antigen, and STn antigen (Arike et al., 2017; Brazil & Parkos, 2022; Consortium, 2022; Johansson & Hansson, 2016; Jumper et al., 2021; Tunyasuvunakool et al., 2021).

Transmembrane mucins, such as MUC1, MUC3, MUC4, and MUC17, are components of the glycocalyx (Tailford et al., 2015). Secreted mucins form the mucus layer, and the MUC2 glycoprotein is the major mucin that forms this layer in the colon and small intestine (Johansson et al., 2008). The colonic mucus has two layers (a denser inner layer that is largely free of bacteria and a looser outer layer that contains some bacteria); the small intestine has a mucus structure composed of only one layer (Figures 1 and 2) (Eloe-Fadrosh & Rasko, 2013). The interplay between the gut mucins and the gut microbiota is essential to establish a healthy mucus layer, as mucin glycosylation shapes the nature and variety of mucus and contributes to the gut microbiota diversity and function (Etienne-Mesmin et al., 2019; Juge, 2012). The mucus layer contains many attachment sites, allowing it to be a habitat for bacteria (Juge, 2012). Impairment of the mucus layer grants the bacteria access to the epithelial layer, which can cause inflammation (Figure 2b) (Johansson et al., 2014).

4.1.2 |. Mucin glycosylation and dysbiosis/disease

The glycan expression changes from infancy to adulthood, and similar to how the gut microbiota differs among individuals, mucin glycans also differ (Bergstrom & Xia, 2013; Derrien et al., 2010; Naughton et al., 2013). Genetic factors play a significant role in these differences as fucosylation of oligosaccharides depends on fucosyltransferase (FUT) secretor status and Lewis genes (Naughton et al., 2013). FUT is the enzyme that adds Fuc to epithelial glycan chains in conformations of α1–2, α1–3, or α1–6 (Kudelka et al., 2020). It has been demonstrated that α1–2 FUT-2 (FUT2) polymorphism is highly associated with IBD susceptibility, and individuals with FUT2 non-secretor status (inactivating polymorphisms of FUT2) have an increased risk for Crohn’s disease as this deficiency inhibits Notch signaling, and possesses different microbiota properties than secreter status individuals (Table 1) (McGovern et al., 2010; Rausch et al., 2011; Wang, Huang, et al., 2017). The addition of α1–2 Fuc to terminal Gal residues forms the H antigen (Fucα1–2Gal). FUT2 encodes the H antigen on intestinal epithelial cells, which allows for bacterial binding such as H. pylori (Brazil & Parkos, 2022; Pacheco et al., 2012; Varki, 2017). Increased epithelial α1–2 Fuc expression also helps promote the colonization of commensal bacteria like Bacteroides and Ruminococcaceae and, at the same time, reduce the colonization of opportunistic gut bacteria like Enterococcus faecalis (Figure 2a) (Pacheco et al., 2012). There are many aspects to how fucosylation mediates this homeostatic gut environment. Epithelial fucosylation can be negatively affected by IL-10-producing CD4⁺ T cells (Figure 2b) (Goto et al., 2015). On the other hand, commensal and pathogenic bacteria (and their products like LPS) stimulate group 3 innate lymphoid cells (ILC3s), producing IL-22 and inducing α1–2 fucosylation of intraepithelial cells (Dias et al., 2018).

Mucin glycans provide surfaces for bacteria to anchor themselves (Derrien et al., 2010; Naughton et al., 2013). Select gut bacterial populations encode and use enzymes such as glycoside hydrolases and proteases specific for glycan removal and processing of mucins, giving access to the anchor attachment points (Derrien et al., 2010; Fang et al., 2021; Naughton et al., 2013; Tailford et al., 2015). Mucin glycans act like decoys for epithelial surface glycans and confine bacteria to the mucus layer, preventing them from accessing epithelial surface glycans (Werlang et al., 2019). However, certain bacteria such as B. fragilis can directly attach to epithelial mucins, highlighting the significance of glycans in the diversity of gut microbiota (Glowacki & Martens, 2021; Huang et al., 2011). Accordingly, the mucus layer in the gut protects the epithelial layer and helps prevent microbial invasion by separating microbes from the intestinal surface (Figure 2a) (Johansson et al., 2008). This process helps control immune activation and maintains the balance in the host-microbial relationship (Johansson et al., 2008). However, pathogens may colonize the GI tract by binding to the fucosylated mucin glycans (Lee et al., 2022). For instance, Salmonella typhimurium Std fimbriae bind to these glycans to adhere to the gut (Suwandi et al., 2019).

Mucin production results in a continuous flow of mucus, which can change during inflammation (Table 1) (Antoni et al., 2014; McLoughlin et al., 2016; Sicard et al., 2017). While Crohn’s disease is associated with increased mucus production, the mucus layer is thinner and discontinuous in ulcerative colitis, with mucin glycosylation altered to shorter and less complex glycoforms (Figure 3b) (Dorofeyev et al., 2013; Singh et al., 2022). The properties of MUC2 and other mucins vary with the disease course, activity, and severity (Larsson et al., 2011; Pullan et al., 1994). Impairment of the mucus barrier results in increased permeability, thereby allowing easier access for bacteria to the epithelial layer, and consequently inflammation (Johansson et al., 2008). Decreased O-glycosylation of mucin may cause faster digestion by bacteria—consequently, the mucus barrier malfunctions, increasing the susceptibility to diseases like IBDs (An et al., 2007; Fu et al., 2011). A healthy gut microbiota helps maintain the integrity of mucus by preventing dysbiosis-induced changes to MUC2 production and thickness (Wlodarska et al., 2011).

The MUC2 monomer contains more than 5000 amino acids, rich in proline, serine, and threonine (Figure 3a) (Consortium, 2022; Godl et al., 2002; Svensson et al., 2018). It regulates the gut microbiota by providing nutrients, acting as ligands to microbial agents, and mediating host signaling (Bansil & Turner, 2018; Kudelka et al., 2016). A functional mucus layer cannot form without MUC2, as demonstrated by the development of bacterial overgrowth, spontaneous colitis, and progressive carcinomas in MUC2-deficient mice (Johansson et al., 2008; Van der Sluis et al., 2006). Like complete loss of MUC2, reduced MUC2 expression or MUC2 mutations caused spontaneous colitis in mice (Heazlewood et al., 2008; Van der Sluis et al., 2006). Bacterial products like LPS, lipoteichoic acids, and flagellin can activate the expression of MUC2 via TLRs and trigger mucin secretion from goblet cells (Birchenough et al., 2016; Dharmani et al., 2009; Hayashi et al., 2001). Germ-free mice show a decreased MUC2 expression and impaired mucosal layer due to their fewer and smaller goblet cells and less sialylated glycans in the mucus layer (Table 1) (Arike et al., 2017; Johansson et al., 2015).

The resident microbiota can affect the function of goblet cells and, thereby, the mucus layer properties via the release of bioactive compounds (Deplancke & Gaskins, 2001). In addition, a gram-positive bacterium, Lactobacillus plantarum (L. plantarum), has been shown to increase the secretion of MUC2 and MUC3 (Mack et al., 2003). These commensal bacteria not only stimulate the secretion of different mucin types but also play an essential role in preventing pathogenic bacteria from gaining access to the epithelial layer. For instance, increased expression of MUC3 can inhibit the attachment of enteropathogenic Escherichia coli (EPEC) (Mack et al., 1999). In addition, a combination of probiotic bacteria Lactobacillus and Bifidobacterium spp. attenuates the pathogenicity of C. jejuni by stimulating the production of unique (i.e., low luminal pH) mucus layers (Alemka et al., 2010; Moran et al., 2011).

Mucin properties are also altered in CRC patients, as it was demonstrated that MUC1 expression is increased in these patients (Table 1) (Lau et al., 2004). MUC1 is hyperglycosylated and expressed at low levels in the colonic tissue of healthy individuals (up to 10%); conversely, it is hypoglycosylated and expressed in very high levels in the colonic tissue of CRC patients (Lau et al., 2004; Limburg et al., 2000). In addition, increased MUC1 was associated with poor prognosis and metastasis (Xu et al., 2015). Additionally, MUC2 expression levels decreased in patients with non-mucinous colon adenocarcinomas (Wang, Jin, et al., 2017). Alterations of MUC2 glycosylation and increases in tumor-associated carbohydrate antigens (TACAs) such as Tn antigen and sialyl Tn (STn) antigen were observed in both UC and CRC patients (Larsson et al., 2011; Sun et al., 2018). MUC5AC, a mucin expressed in gastric mucus and absent in the colon, was found to be expressed in CRCs (Rico et al., 2021).

Mucins constitute a significant source of sialic acid, and Neu5Ac is the most abundant sialic acid in the GI system. In adults, while the Fuc expression decreases from the proximal to the distal gut, sialic acid expression increases from the ileum to the colon (Bell et al., 2023; Robbe et al., 2003). A recent human study has demonstrated that terminal sialylation of mucin glycans by ST6GalNAcT plays an essential role in the integrity of the mucus layer by preventing excessive bacterial proteolytic degradation (Yao et al., 2022). Furthermore, mutations of ST6 cause a defective mucus layer in patients with IBDs (Yao et al., 2022). Bacterial sialidases can liberate the sialic acids that cap mucin glycans for use by the same bacteria, other commensal bacteria, or pathogenic bacteria (Juge et al., 2016). Besides the sialylation of mucin, sialylation of IgG also plays a part in the IBD pathogenesis as serum IgG sialylation levels decrease in patients with ulcerative colitis and Crohn’s disease (Šimurina et al., 2018).

4.2 |. Bacterial processing of glycans

Dietary carbohydrates are essential for the gut microbiota (Martens et al., 2008). Many gut commensals have carbohydrate-active enzymes (CAZymes), which facilitate the processing of a range of glycans (Figure 2c) (Lozupone et al., 2008; Pereira & Berry, 2017). CAZymes, encoded by bacteria, vary among individuals depending on factors such as age and geography, due to their effects on gut microbiota (Bhattacharya et al., 2015). Differential glycan preferences of gut microbes result in diverse metabolism (Pereira & Berry, 2017). For example, B. thetaiotaomicron, B. fragilis, and Ruminococcus torques can break down molecules such as mucin (Huang et al., 2011; Martens et al., 2009; Png et al., 2010). B. thetaiotaomicron can metabolize L-fucose as an energy source and induce host FUTs to increase mucosal fucosylation, creating a beneficial environment for itself (Bry et al., 1996; Hooper et al., 1999). B. thetaiotaomicron increases sialic acid expression (Wrzosek et al., 2013). B. thetaiotaomicron and Ruminococcus gnavus encode exoglycosidases, such as fucosidases and sialidases, each releasing distinct glycoforms from mucin for their use as energy source (Bell et al., 2019; Luis et al., 2021; Pacheco et al., 2012). In addition, B. thetaiotaomicron encodes for endoglycosidases of the Glycoside Hydrolase Family 16 (GH16) that are upregulated during their growth on mucin layer (Crouch et al., 2020).

Due to its high glycan content, the mucus layer serves as a nutrient source for the inhabitant bacteria (Lozupone et al., 2008). Mucin glycan utilization gives bacteria a consistent nutrient supply and helps the bacteria colonize the mucus layer (Marcobal et al., 2013). In the hGM, due to their diverse CAZymes, Bacteroidetes are general glycan degraders that can use both dietary and host glycans (Ndeh & Gilbert, 2018). Bifidobacteria and Firmicutes genera show similar glycan degradation patterns (Ndeh & Gilbert, 2018). Akkermansia muciniphila is another important bacterial species that degrades mucus glycans into acetate to support butyrate-producing bacteria (Paone & Cani, 2020; Shin et al., 2019). Conversely, A. muciniphila can show mucus thinning effects in low-fiber diets and dysbiosis (Figure 2d) (Yoshihara et al., 2020). Mucin-degrading bacteria can also generate SCFAs through fermentation. These SCFAs can be utilized by non-mucolytic bacteria or the host to recover the energy used in mucin synthesis and secretion (Breugelmans et al., 2022). MUC2 expression can also be increased by SCFAs (Burger-van Paassen et al., 2009).

Mucin-glycan degradation by bacteria is essential in establishing a stable microbiota (Sicard et al., 2017). The symbiotic relationship between the host and the microbiota relies on the microbes’ capabilities of host glycan digestion and the host’s ability to secrete mucin glycans for microbial stimuli (Koropatkin et al., 2012). If the dietary polysaccharides were to be depleted from the host’s diet, this would result in a significant shift to gut bacterial consumption of host mucus (Sonnenburg et al., 2005). If the host has a low-fiber diet, this results in decreased microbial diversity and a change in microbial composition to have more reliance on host mucus (Figure 2d) (Johansson et al., 2014; Sonnenburg et al., 2005). Increased reliance on host mucus eventually wears down the mucus barrier and causes an inevitable breach of the mucus layer, as observed in ulcerative colitis patients (Johansson et al., 2014). This data also supports the finding that the low fiber content in the Western diet leads to increased IBD prevalence (Johansson et al., 2014). Increased abundance of mucin-degrading bacteria in the context of a fiber-deficient diet has also been shown to increase inflammation and pathogen susceptibility in a mouse model (Desai et al., 2016).

On the other hand, Bifidobacteria can break down human milk oligosaccharides (HMOs) to aid digestion, a process necessary for infants who lack the enzymatic capability to process HMOs (Bondue et al., 2016; Özcan & Sela, 2018). Exposure to HMOs in breast milk in infancy helps Bifidobacteria to colonize the gut and help the host develop an immune tolerance towards commensal bacteria since HMOs act as a carbon source for the gut microbiota and host (Lawson et al., 2020). HMO metabolism is a virulence-suppressing process and is required to prevent the adhesion of pathogens to the intestinal epithelia as HMOs resemble epithelial surface glycans, allowing them to act as decoy receptors for bacteria (like E. coli and Vibrio cholerae), and hence, preventing the attachment of the bacteria to the gut (Bondue et al., 2016; Kulinich & Liu, 2016). HMOs assert their anti-inflammatory effects by regulating interleukin production and lymphocyte activation. Additionally, sialylated HMOs can help maintain a Th1/Th2 balance (Chleilat et al., 2020; Eiwegger et al., 2010).

The process of dietary fiber fermentation by the gut microbiota starts with the breakdown of complex glycans into simpler sugars, which are then fermented by the intestinal anaerobic microorganisms (Figure 2c). This fermentation process leads to the production of short-chain fatty acids (SCFAs), which are absorbed from the colon to be used in numerous metabolic processes (den Besten et al., 2013; Morrison & Preston, 2016). One such SCFA is butyrate. It is the preferred energy source of colonocytes, facilitating the maturation of the colonic mucus barrier (Beaumont et al., 2020; Litvak et al., 2018).

4.3 |. Bacterial glycans

Bacterial glycans are essential in bacterial colonization, host tissue invasion, and immune response modulation in the gut (Prado Acosta & Lepenies, 2019). Glycoconjugates produced by bacteria include LPS, teichoic acids, glycoproteins, glycolipids, peptidoglycans, and capsular polysaccharides (Tan et al., 2015). Some species, such as Pseudomonas aeruginosa and Neisseria meningitidis, express O-linked glycoproteins, mainly found in pilin and flagellin subunits; other species, such as Haemophilus influenzae express N-linked glycoproteins (Castric, 1995; Ku et al., 2009). L. plantarum, a commensal bacterium, expresses an O-glycosylated protein, Acm2, as its primary autolysin (Latousakis & Juge, 2018). The glycosylation machinery in C. jejuni mainly relies on oligosaccharyltransferase PglB, which transfers oligosaccharides to proteins (Linton et al., 2005). C. jejuni deficient in PglB have attenuated pathogenesis, highlighting the microbe’s reliance on its glycosylation system (Abouelhadid et al., 2019).

Bacterial surface glycans can be recognized by immune cells (Adibekian et al., 2011). This recognition may result in the engulfment of the microbe by DCs, allowing for the processing and presentation of immunogenic epitopes (Kerrigan & Brown, 2009; Paschall et al., 2023; Rabinovich & Croci, 2012). Zwitterionic polysaccharide (ZPS) capsules in bacteria are highly studied immunomodulatory bacterial glycans, which play many essential roles in regulating intestinal immune homeostasis (Avci & Kasper, 2010). For example, Polysaccharide A (PSA) of B. fragilis, a ZPS, induces the production of IL-10 through APCs, creating a tolerogenic environment to colonize (Round & Mazmanian, 2010). It can also skew the T-helper balance in favor of Th1 and help maintain the Th1/Th2 balance in the gut (Mazmanian et al., 2005). Bacteria can also mimic host glycan structures (molecular mimicry) (Geijtenbeek & Gringhuis, 2009; Kudelka et al., 2020). Bacteria like B. fragilis can use the free fucose on their capsule and imitate the fucosylated epithelial glycans (Coyne et al., 2005). This process also aids in immune evasion of the bacteria (Kudelka et al., 2020).

Apart from expressing glycans on their surface, gut bacteria also produce enzymes that modulate glycan expression on immune cells (Paschall et al., 2023). For example, immune evasion by Streptococcus pyogenes (Group A streptococcus), a pathogen that causes diseases like acute rheumatic fever and post-streptococcal glomerulonephritis, is attained by direct glycan modulation on antibodies (Naegeli et al., 2019; Walker et al., 2014). A targeted mass spectrometry study has shown that this is achieved by an endoglycosidase, EndoS, that cleaves the conserved N-glycan on IgG antibodies (Naegeli et al., 2019).

5 |. CONCLUDING REMARKS AND FUTURE DIRECTIONS

The human gut is vast and complex, posing a challenge in comprehending the physiological processes that regulate homeostasis. Past and ongoing research studies continue to provide important insights into its intricacies. Our evolving understanding of the hGM leads to new investigations on elucidating dysbiosis-associated disease mechanisms such as IBDs and CRCs. In these GI tract-associated diseases manifested by inflammation—in addition to the already established pathophysiological mechanisms—the invasion of the GI tract by the pathogenic bacteria may play an inducing role.

Glycans regulate immune cells, maintain mucus structure and integrity, and promote symbiosis among gut microbes. Aberrant mucin O-glycosylation and overexpression of hypoglycosylated glycoforms such as T, Tn, and STn antigens are observed in both CRC and IBD tissues (Figure 3b) (Campbell et al., 1995; Kudelka et al., 2020). The mechanisms behind the glycosylation changes in ulcerative colitis and CRCs are yet to be fully elucidated. As a result, it is becoming evident that demystifying specific mechanisms by which glycans contribute to regulatory processes in the GI tract would lead to developing effective treatments that modify the gut glycome, promote homeostasis, and prevent diseases.

DATA AVAILABILITY STATEMENT

Data sharing is not applicable to this article as no new data were created or analyzed in this study.