A sticky end for gastrointestinal helminths; the role of the mucus barrier

Summary Gastrointestinal (GI) nematodes are a group of successful multicellular parasites that have evolved to coexist within the intestinal niche of multiple species. It is estimated that over 10% of the world's population are chronically infected by GI nematodes, making this group of parasitic nematodes a major burden to global health. Despite the large number of affected individuals, there are few effective treatments to eradicate these infections. Research into GI nematode infections has primarily focused on defining the immunological and pathological consequences on host protection. One important but neglected aspect of host protection is mucus, and the concept that mucus is just a simple barrier is no longer tenable. In fact, mucus is a highly regulated and dynamic‐secreted matrix, underpinned by a physical hydrated network of highly glycosylated mucins, which is increasingly recognized to have a key protective role against GI nematode infections. Unravelling the complex interplay between mucins, the underlying epithelium and immune cells during infection are a major challenge and are required to fully define the protective role of the mucus barrier. This review summarizes the current state of knowledge on mucins and the mucus barrier during GI nematode infections, with particular focus on murine models of infection.


| INTRODUCTION
Intestinal nematodes are among the most common and widely distributed animal parasites of humans, estimated to infect over 2.5 billion of the world's population, the majority of infections occurring in children. 1,2 Among the most prevalent intestinal worms are the hookworm (Ancylostoma duodenale and Necator americanus), roundworm (Ascaris lumbricoides) and whipworm (Trichuris trichiura), which are typically found endemic in developing and tropical countries. These infections are normally transmitted by soil and are chronic in nature, which is in part due to endemic regions often lacking intervention that can curb transmission (ie medicinal care, diagnosis tools, effective sanitation, protocols to prevent reinfection and efficient treatment plans). 3 Globally, these infections are accountable for causing severe morbidity to over 300 million individuals. 4 Clinical manifestations of infections include malnutrition, cognitive dysfunction, vitamin deficiencies and growth retardation, 1,4 which all severely impair the quality of life of affected individuals. Despite their prevalence, this group of parasitic infections is considered as "minor" and often neglected in clinical treatment.
Current research is focused on defining host-protective responses that lead to parasite expulsion, which are exceedingly difficult to elucidate within infected human populations. However, studies using various well-established laboratory models of GI nematode infections have greatly contributed to our knowledge in understating how the host coordinates immune responses associated with resistance.

Summary
Gastrointestinal (GI) nematodes are a group of successful multicellular parasites that have evolved to coexist within the intestinal niche of multiple species. It is estimated that over 10% of the world's population are chronically infected by GI nematodes, making this group of parasitic nematodes a major burden to global health. Despite the large number of affected individuals, there are few effective treatments to eradicate these infections. Research into GI nematode infections has primarily focused on defining the immunological and pathological consequences on host protection. One important but neglected aspect of host protection is mucus, and the concept that mucus is just a simple barrier is no longer tenable. In fact, mucus is a highly regulated and dynamic-secreted matrix, underpinned by a physical hydrated network of highly glycosylated mucins, which is increasingly recognized to have a key protective role against GI nematode infections. Unravelling the complex interplay between mucins, the underlying epithelium and immune cells during infection are a major challenge and are required to fully define the protective role of the mucus barrier. This review summarizes the current state of knowledge on mucins and the mucus barrier during GI nematode infections, with particular focus on murine models of infection.

K E Y W O R D S
goblet cell, innate immunity, mucosal immunity, Nippostrongylus brasiliensis, Trichinella spiralis, Trichuris spp Perhaps, the most commonly used murine models of helminth infections include Trichuris muris, Trichinella spiralis, Nippostronglylus brasiliensis and Heligmosomoides polygyrus, and a summary of each parasites' life cycle during infection is shown in Table 1.
Typically, most laboratory models of intestinal helminth infections can elicit a strong CD4 + Th2-mediated immune response. Immune characteristics associated with a Th2 mediated environment are the secretion of type 2 signature cytokines (IL-4, IL-13, IL-9 and IL-5), activation of Th2 cells, antibody class switching to IgG1 (in mice) and IgE, and induction of alternatively activated macrophages, eosinophils, basophils and mast cells. 5 This response is often referred to as an "allergic" immune response and is associated with goblet cell hyperplasia.
As goblet cells are the major source of mucins (the major macromolecule of the intestinal mucus barrier), the expansion of this cell type can lead to the increased secretion of mucins which can consequently lead to alterations in the protective properties of the mucus barrier. The altered barrier can directly or indirectly affect parasite establishment within the GI niche, thus impeding the ability of the parasite to productively interact with the host and to thrive and survive. The involvement of mucus as a protective barrier during GI nematode infection was initially identified in the early 1980s, whereby the "mucus-trap" hypothesis was coined. [6][7][8] It was demonstrated that during T. spiralis and N. brasiliensis infection, the parasites were surrounded by mucus prior to their expulsion, indicating a role for mucus to physically separate and prevent the establishment of parasites within their niche.
This observation suggested a direct role for the mucus barrier as an effector mechanism to protect the host and aid parasite expulsion.
Indeed, subsequent characterization of animal models for GI helminth infections and the development of protocols to assess mucosal barrier properties have allowed the development of robust systems to directly investigate aspects of mucus barrier function and properties in vivo.
These studies have demonstrated that mucins and mucus-associated proteins hold key roles in altering the intestinal niche to enhance parasite expulsion, thus contributing to immune-mediated host protection. [9][10][11] Further insight into the precise functional role(s) that mucins and mucus-associated proteins play within the mucus barrier may uncover potential avenues for novel therapeutic targets to eradicate this group of important neglected tropical diseases.
In this short review, we discuss the nature and formation of the intestinal mucus barrier and its mucin components during homeostasis. We will provide details on how mucins form mucus and describe the complexities of mucin synthesis, structure and function. Furthermore, we will elaborate how the immune system controls mucin production and properties to produce a mucus barrier with effective host-protective function to combat GI nematode infections. Together this will highlight that mucus is not just a passive physical barrier but is a highly regulated and dynamic defence mechanism, and an important part of a coordinated immune-driven host response against GI nematode infections.

| THE INTESTINAL MUCUS BARRIER
The mucosa of the intestine is made up of a monolayer of cells arranged in multiple crypts that physically separates the external environment and subepithelium. The apical surface of the intestinal mucosal cells is protected by a carbohydrate-rich barrier comprised of the cell-tethered glycocalyx and the overlaying mucus gel; major macromolecular constituents of both components of the barrier are the O-linked glycoproteins known as mucins. To aid site-specific roles in the intestine, the mucus barrier is selectively organized in different regions of the GI tract and increases in thickness along its length; measurements in rats show the barrier is thickest in the colon (~830 μm) and thinnest in the jejunum (~123 μm). 12 The small intestine has a single layer of mucus to facilitate the transition of nutrients for dietary absorption, whereas the colon has a thicker and more highly organized two-tiered mucus barrier, composed of a firmly adherent inner layer (~50 μm) and a loose outer layer (~100 μm), [12][13][14] and this organization has recently been shown to be affected by the faecal load 15 (Figure 1). The mucus barrier architecture is required to maintain the large number of bacterial species colonizing the colon to aid symbiosis but prevent bacterial infiltration to the epithelium. 16 Although the function of mucus has historically been accepted to act only as a physical barrier, it is now well-recognized that it has other T A B L E 1 Commonly used murine gastrointestinal (GI) colonizing nematodes, describing the niche and life cycle of parasites

Murine GI nematode Type of parasite Life cycle GI niche
Trichuris muris Whipworm After ingestion of embryonated eggs, they hatch and invade the epithelial layer of the caecum and proximal colon, undergoing 4 moults before becoming adults.

Caecum
Trichinella spiralis Roundworm Infection occurs via ingestion of L1 larvae found within the muscle of a previously infected host. L1 larvae invade epithelial cells of the small intestine where they rapidly moult to adulthood.

Nippostrongylus brasiliensis
Hookworm L3 larvae penetrate the skin, pass through the vasculature to the airways and crawl up the bronchi to be swallowed into the GI tract where they inhabit the small intestine.

Heligmosomoides polygyrus
Roundworm/hookworm Free-living L3 larvae are ingested and penetrate the submucosa of the small intestine; they moult and then reemerge into the intestinal lumen of the small intestine remaining in the villi.
Small intestine general intrinsic roles such as providing specific ligands for pathogen entrapment, lubrication, hydration and aiding digestion. 17 However, exactly how the intestinal mucus barrier is organized and assembled is not fully defined, but the gel-like properties of mucus are primarily dictated by the unique structure of the polymeric gel-forming mucin, MUC2 (humans)/Muc2 (mice).

| MUCINS
Mucins are a family of large and highly O-glycosylated proteins that 80% of the mass accounted for by glycans. 32 These glycan chains are highly heterogeneous in chain length and composition even at homeostasis. Importantly, glycan structure can be influenced during parasitic infections, which aids host protection against pathogenesis (which will be discussed further below). [33][34][35][36] Finally, MUC2 dimers have been proposed to trimerize through disulphide bonds mediated by cysteine residues located in the Nterminal vWfD3 domain and isopeptide bonds formed between the side chains of lysine and glutamine residues (Figure 3, step 5). 27 These covalent linkages give rise to very large and highly glyco-

| IMMUNE CONTROL AGAINST GI NEMATODE INFECTION
Infection with GI nematodes is commonly associated with the generation of type 2 immunity; the cytokine IL-13 is a critical driver for this response and is primarily derived from type 2 innate lymphoid cells (ILC2) and Th2 cells. The induction of IL-13 and IL-4 leads to the expansion of goblet cells, a trait that has been observed during N. brasiliensis, T. spiralis and acute T. muris infections. 10,49,50 As goblet cells are the major mucin-producing cells in the intestine, this expansion leads to alterations in mucus barrier properties, through secretion of mucins and other goblet cell-associated proteins. In recent years, there has been significant progress in understanding the initial mediators of goblet cell hyperplasia during GI nematode infection.
ILC2s were originally identified as an alternative source of type 2 cytokines in mice lacking T or B cells 51 70 These findings suggest that mucus is a crucial innate defence mechanism against invading GI parasitic nematodes and the known roles of different components of the mucus barrier are discussed below.

| GI NEMATODE INFECTION AND MUCINS
As  73 Further studies showed there was an induction of the polymeric mucin Muc5ac, normally a gastric and lung mucin, which was important for T. muris parasite expulsion and suggested a protective function for this mucin in the intestine. 9 This was confirmed using Muc5ac-deficient mice, which were completely susceptible to T. muris infection unlike their wild-type counterparts. Importantly, the susceptible phenotype was not reversed even after administration of anti-IFNγ to skew the Th1 dominated environment generated in chronic infection towards a Th2-directed immune response, normally associated with resistance. 10 Importantly, there was an induction of Muc5ac transcripts in T. suis infected pigs, suggesting a protective role across species, 74 and there is data to suggest that Muc5ac may also have a broad antihelminth action as Muc5ac null mice were also impaired in their ability to efficiently expel N. brasiliensis and T. spiralis. 9 The invading GI nematodes are likely, therefore, to employ strat- After helminth infection tuft cells will secrete IL-25, which leads to the production of type 2 cytokines, predominately ILC2 derived IL-13, which together with Th2 derived IL-13 leads to goblet cell (GC) and tuft cell hyperplasia and changes in mucin production (Muc2 and Muc5ac) and mucin properties (glycosylation). These alterations change the properties of the mucus barrier to aid host protection against intestinal nematode infection significant upregulation of serine protease inhibitors (ie serpins) that protect mucin polymers from degradation maintaining the integrity of the mucus barrier. 35 Further work is required to elucidate the signalling cues employed to initiate Muc5ac expression within the intestine, as this pathway could be a potential therapeutic target to induce parasite expulsion in humans and domestic animals.

| GI NEMATODE INFECTION AND ALTERATIONS IN MUCIN GLYCOME
A unique feature of mucins is the heterogeneous array of glycan structures that decorate the polypeptide backbone, and these glycans have well-established roles in influencing pathogenic organisms, including GI nematodes. 10 75 Yamauchi et al 77  Furthermore, Dolichos biflorus agglutinin (DBA) and Muc2 dual staining showed a higher prevalence of GalNAc residues on Muc2 in acute T. muris infection compared to chronic infection. 10 The functional consequences of these changes remain to be elucidated.
One aspect of mucin glycosylation that has received most atten-  75 Furthermore, in a murine model with reduced mucin sulphation at homeostasis due to a genetic deletion of sulphate anion transporter 1 (Sat-1), mice that would normally be resistant to infection become susceptible, despite the prevailing Th2 immune response. 75 These changes in mucin glycosylation lead to global changes in the mucosal barrier, affecting mucin charge density, which leads to direct alterations within the barrier, including mucus hydration and viscosity that in turn may hinder the parasites' ability to degrade the mucus and thus contribute to host protection. However, it is important to note that as the intestinal tract is such a dynamic and exposed tissue, it is also likely that the mucus gel will contain exfoliated cells from the rapidly turned over epithelial layer, RELMβ is found within ceacal and colonic mucus as a hexamer and trimer and is induced by a Th2 response. 94 It has been proposed that RELMβ can affect the ATP levels and hence the fitness of H. polygyrus and N. brasiliensis through impairing the parasites ability to feed.

| GI NEMATODE INFECTION AND MUCUS-ASSOCIATED PROTEINS
Moreover, RELMβ can aid host protection against N. brasiliensis by causing entrapment of the parasite and reducing parasite motility. 88 However, during T. muris and T. spiralis infection there is an induction of RELMβ expression, but it appears to play little role in expulsion. 87 Additionally, the expression of antimicrobial agents derived from goblet cells, namely angiogenin 4, intelectin-1 and intelectin-3, have been associated with T. muris expulsion, but their functional importance has yet to be determined. 89,90,95 Trefoil factors (TFFs) are a family of 3 cysteine-rich proteins, TFF1, TFF2 and TFF3, having roles in mucosal repair and protection against GI insult. 96 Studies have demonstrated that TFFs interact with mucins to aid mucus gel integrity. 92 For example, TFF3 has been demonstrated to promote mucosal barrier protection during a rat colitis model. 97 Despite this, T. spiralis-infected TFF3 knockout mice had no clear phenotype in comparison with their wild-type counterpart during infection. 91 It has been suggested, however, that TFF2 plays important roles during the lung stage of N. brasiliensis infection, which has been correlated with augmenting both IL-33 and Muc5ac expressions within the lung. 98

| CONCLUSION
Taken together, these data suggest that goblet cells and their secreted products, in particular the polymeric mucins, are important elements for initial protection against GI helminths, and necessary for subsequent clearance of parasites during infection. Not only is there a change in mucus barrier composition and properties, but there is also changes in mucin expression and glycosylation during GI nematode infections. These changes in the mucus barrier constitute a coordinated and critical arm of the innate immune effector response against GI helminths. A better understanding of the regulatory pathways involved in eliciting these changes could highlight novel therapeutic targets to help eradicate this prevalent group of parasites.