University of Groningen
Modulating the immune system and intestinal barrier integrity with functional oligosaccharides
Figueroa Lozano, Susana
DOI:
10.33612/diss.151659844
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Publication date:
2021
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Figueroa Lozano, S. (2021). Modulating the immune system and intestinal barrier integrity with functional
oligosaccharides. University of Groningen. https://doi.org/10.33612/diss.151659844
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General discussion
The carbohydrate fraction in human milk is rich in free and conjugated oligosaccharides. The structural diversity of this fraction is essential for the immune and metabolic development in newborns [1]. The accumulated knowledge about human milk composition has assisted infant formula evolution. In recent years the efforts to improve the functionality of human-milk substitutes have transitioned from mimicking nutritional and caloric value to simulating the beneficial effects of human milk. To emulate human milk immune functionality, infant formula is supplemented among others with functional oligosaccharides, such as bovine lactoferrin (bLF), galacto-oligosaccharides (GOS), and 2’-fucosyllactose (2’-FL). Because these compounds are resistant to intestinal digestion, they serve as growth substrate for the intestinal microbiota [2–4]. Besides this microbiota-dependent effect on the immune system, they are used because they exhibit crucial direct effects on the intestinal homeostasis. Bovine LF, GOS, and 2’-FL are currently used as ingredients in the food industry and are considered safe for infant nutrition.
More recently, it has been documented that these compounds have a broad spectrum of biological activities because they influence the human glycome due to their structural composition. The human glycome is defined as the repertoire of glycans and glycoconjugates produced by a cell under specific conditions of time, environment, and location. This glycome is shaped both by genetic and environmental factors, such as diet. Therefore, variations in the content of mannose, sialic acid, glucose, galactose, fucose, and glucosamine present in dietary carbohydrates, change the composition of the glycome. Although the specific mechanism involved are not well known, this translates for instance in the modulation of the immune response and the intestinal barrier function.
Therefore, the aim of this thesis was to investigate in vitro how bLF, GOS, and 2’-FL modulate the function of the intestinal barrier function by affecting epithelial and immune cells. First, in the case of bLF, the composition of the conjugated glycans determines physicochemical characteristics and contributes to the biological activity of the protein. By modifying the chemical composition of the bLF glycans, we studied the interaction of these modified and unmodified structures derived from bLF with innate immune receptors known as pattern recognition receptors (PRRs). Second, we explored the effect of GOS content of transgalactosylated oligosaccharides, lactose, and glycosidic linkage in the modulation of the secretory function of goblet cells. Finally, we assessed the impact of enzymatically produced 2’-FL on the function of goblet cells under homeostatic conditions and the effects on the intestinal barrier integrity upon disruption with a calcium ionophore.
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Mannose and sialic acid content on bLF glycans shape the immunomodulatory
properties of the glycoprotein
In chapter 2, we assessed how the changes in the glycan composition, namely the content of mannose or sialic acid on the glycans chains of bLF and the glycans isolated from it, had an effect on the immune response. To alter the content of mannose and sialic acid on the glycans, bLF was treated either with mannosidase or neuroamidase. Our findings showed that the glycan chain composition either conjugated or isolated from the protein core is relevant for the direct bioactivity of bLF mediated by Toll-like receptors (TLRs).
While most research on bLF function focus on the impact of the protein core, we studied the effects of the covalently attached glycans (chapter 2 and 3). Although human and bLF share 77% protein homology, there is a substantial difference in the N-glycan composition of both glycoproteins. The advances in highly sensitive approaches to characterise these structural make-up differences, have enabled the identification of new structures and the quantification of the changes in composition during time [5]. As a result, it has been become possible to understand the chemical determinants that play a role in function-structure relationships of glycoproteins and isolated glycans. For instance, while the mannosylation of bLF prevents the adhesion of E. Coli [6], the sialic acid present on the glycans chains of bLF is believed to be the main factor that impairs Helicobacter
P. colonization in a mouse model [7]. The results from chapter 2 indicate that the
composition of the bLF glycans affect directly the immune response mediated by TLRs. The most important aspect of this immunomodulation is that changes in the composition of the glycoprotein glycans can skew the immune response towards a pro- or anti-inflammatory pathways. The understanding of the impact of glycosylation of proteins has broaden the knowledge about disease onset and progression [8]. For instance, sialylation has been found to be key for the anti-inflammatory activity of intravenous gamma globulin [9]. Although it is unknown how this changes in sialylation occur specifically, the expression of sialic acid on proteins or cells has been linked to the dietary intake of a specific type of sialic acid that is present in bovine meat but absent in humans. This sialic acid known as
N-glycolylneuraminic acid (Neu5Gc) can be incorporated in different human glycoproteins
since early stages, given that they are contained as well in infant formula. The content of Neu5Gc in bLF is approximately 8.5% [10]. The presence of this member of the sialic acid in the human glycome has been associated with exacerbation in inflammation and autoimmune disease [11]. In chapter 2, the reduction in sialic acid diminished the release of NF-κB but also increased the inhibitory capacity of isolated N-glycans (chapter 3). Therefore, the possibility to manipulate the glycosylation of bLF can deliver means to treat different states of inflammation.
The glycosylation of human and bovine glycoproteins, such as bLF, is a dynamic process. Glycosylation is a species- and cell/tissue-specific process and varies among individuals and geographical regions [12]. By reducing the mannose content of bLF (chapter 2), we aimed to simulate the colostrum stage, whereas the reduction in sialic acid resemble more the mature milk phase. In bovine milk, also environmental factors, such as season and diet, alter the glycosylation pattern of their milk [13]. Therefore, a big challenge into the understanding of glycosylation is the technological ability to reproduce these dynamic changes, for instance, in infant formula.
As outlined above, functional studies with modified glycans from bLF are not common. An important limitation is the preparation of larger amounts of modified bLF for further studies, for instance, in animal models, and this factor is crucial to observe the attributes of these structural changes in vivo and its implications on immunogenicity. Furthermore, clinical studies with infants are difficult to design because infants cannot be randomized to consume formulas or human milk, given that human milk remains as the gold standard for infant nutrition. Although infant formulas reinforced with bLF are inferior to human milk, they represent a safe option to feed infants. Nonetheless, with this study we provide insight into the relationship between the glycosylation pattern, namely mannose and sialic acid content of the glycans chains of bLF, in its immunomodulating capacity.
In chapter 3, we studied the effects of isolated partially de-mannosylated and partially de-sialylated N-glycans isolated from bLF on the inhibition of ssRNA40 or R848 induced TLR-8 activation. We studied this inhibitory effect in reporter monocytes overexpressing TLR-8 and we assessed whether this inhibition would change the production of cytokines in monocyte-derived dendritic cells (MoDCs). Our findings suggested the inhibitory capacity of the N-glycans might be mediated by the ability of the N-glycans to interact with the TLR-8 and to impair the configuration changes required for the receptor activation. When tested in MoDCs, the N-glycans significantly decreased the production of IL-6. Furthermore, the partially de-sialylated moiety induced a stronger inhibition of the IL-6 production compared with the partially de-mannosylated N-glycans.
N-glycosylation is considered crucial for the function of milk glycoproteins. However, the
specific roles of isolated N-glycans from dietary sources and whether they have biological effects is unclear. A major reason is that glycosylation is a non-driven-template process. This means that the composition of the glycan chains is not determined genetically but rather influenced by environmental factors that produce glycoproteins with a heterogeneous glycan composition. The separation of diverse glycoforms is troublesome and it is considered an obstacle to understand the role of glycans in the glycoprotein function [14]. In the case of bLF, the low concentration of bLF in bovine milk and the
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difficulties to isolate amounts in the milligram scale, have hindered the study of these molecules until now, as shown in chapter 2. Thanks to advances in enzymatic cleavage and identification of products with mass spectrometry techniques it has been possible to evaluate the function of N-glycans, for instance, in bacterial growth. N-glycans from bovine milk glycoproteins released using Endo-β-N-acetylglucosaminidases (EndoB1-1) enzymes are used by B. lactis and B. infantis as a carbon source for growth to support the selective growth of gut bacteria [15]. The composition of N-glycans not only have an indirect effect on the intestinal microbial colonization, it has been described to alter the anti-bacterial activity of specific glycoproteins derived from human milk compared to cow milk. The heavy fucosylation of human milk makes it more affective to prevent the adhesion of pathogens to the intestinal mucosa when compared to bovine milk. Interestingly, when human milks is defucosylated, the difference in anti-bacterial activity of both types of milk disappears [16]. This does not mean that the glycoproteins loses its anti-pathogenic effects, it indicates that other carbohydrates that form the glycan, such as sialic acid or mannose, play a role as well in this biological activity.
In chapter 3, we showed that N-glycans directly damper the activation of TLR-8, supporting our hypothesis that the isolated glycans also contribute to the immunomodulatory capacity of bLF and such capacity is linked to the glycan composition. The fact that a differential composition can alter directly the immune response (chapter 2) can be used as strategy to develop new strategies for the formulation and design of functional foods and small molecules drugs with therapeutic effects. This could be the case for the treatment of viral infections. Bovine LF has the capacity to bind to the heparan sulphate proteoglycans of viruses, such as SARS-Cov to inhibit the contact of the viral particle with the host cells [17]. Although the mechanism involved has not been elucidated specifically for this virus, it is known that the removal of sialic acid is involved in the inhibition of rotavirus infection by bLF [18].
It is difficult to predict whether these findings can be observed in the in vivo situation. Human digestion can cleave bLF into antimicrobial peptides but there are no studies reporting the presence of glycoside hydrolases capable of breaking the bond types present in human milk glycoproteins [19]. In adults, apo and holo bLF seem to survive gastric passage because of this lack of gastric glycosidases [20]. Therefore, it seems only some species from the intestinal microbiota can cleave the N-glycans from bLF [21], but the extent of bacterial digestion is unknown [22]. In order to get a better picture of the effects of isolated N-glycans on immune responses, further studies could assess the degree of bLF
N-glycan cleavage by specific microbial communities, such as B. infantis, characterize its
Galactose and glucose covalent binding in lactose derived oligosaccharides and
GOS modulate the intestinal barrier at mucus level
In this thesis we have studied the effects of glycans attached to the protein core (chapter
2), glycans isolated from the original glycoprotein (chapter 3), and next we investigated
how molecules enzymatically derived from lactose can modulate epithelial responses at the mucus level and/or the immune response (chapter 4 and 5). The production of glycans, such as GOS or fucosylated oligosaccharides from engineered bacteria has facilitated the study of these compounds directly on the cellular pathways of epithelial responses. The mucus layer and the barrier integrity are two mechanisms that support the function of the intestinal barrier in vivo. Both mechanisms have been suggested to be modulated by HMOS. Although it is accepted that HMOS can affect the barrier function, it is unknown how they or even more specific oligosaccharides, such as GOS or 2’-FL, can alter goblet and epithelial cell function.
The mucus layer plays a crucial role in the protection of maintenance of the intestinal barrier function. In chapter 4, we studied the effects of GOS with different composition and glycosidic linkages on the function of goblet cells. Our results indicate that the direct effects of GOS on the expression of genes that form the intestinal mucus do not require to be purified to exert a direct biological response and that the glycosidic binding among the galactose units can influence the secretory function of goblet cells.
The importance of the differential modulation of mRNA expression of mucus-related genes is, that beyond reinforcing the physical barrier formed by mucus, they modulate immune tolerogenic responses to food intake. MUC2 impairs the secretion of IL-10, a cytokine that inhibits pro-inflammatory cytokines, such as IL-12 and IFN-γ. By this mechanism, MUC2 mitigates inflammatory responses mediated by CD103+ dendritic cells [23]. In animal models, TFF3 overexpression can induce insulin tolerance in obese mice [24], and RETNLB acts as a CD4+ T chemo-attractant that induces cell proliferation during pathogenic infection [25]. The modulation of goblet cells secretory products is important given because these cells directly deliver high molecular weight luminal antigens to DC in the lamina propria via a process called goblet cell-associated antigen passage [26].
GOS elicit responses similar to HMOS. The study of the structural treats of GOS that influence their function shed light on the importance of these oligosaccharides in the interplay between lumen, mucus, and intestinal epithelial layer. GOS not only serve as forage for microbial communities that produce short chain fatty acids, which in turn influences the composition of the mucus layer. This mucus layer maintains the intestinal bacteria and signals the epithelial layer to produce the compound responsible for the host
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defence. GOS have the potential to alter the gastrointestinal glycome by modulating the expression of sulfomucins. The reduction of sulfonated mucin is associated with increased risk to develop inflammatory bowel disease and colorectal cancer. The induction of genes such as CHST5 and GAL3ST2 in the absence of inflammatory mediators, such as IL-13, is important to target the treatment of patients with colonic inflammatory disease.
Although it was out of the scope of this thesis, GOS has the potential to alter substrate availability and in consequence the ability to change the profile of mucin glycosylation. Probiotics, such as GOS and lactulose, are subject of complex enzymatic modifications in order to add sialic acid on their structure [27]. Our results paved the way to the study of such molecules on goblet cells. The high levels of sialic acid of oligosaccharides present in rodent milk change the colonic gene expression in sialic acid pathways [28].
In chapter 5, we assessed the in vitro effects of enzymatically produced 2’-FL in the secretory function of goblet cells and on the intestinal barrier integrity. The results indicate that HMOS isolated from a Secretor+ Lewis+ donor do not alter significantly the expression of MUC2, TFF3, RETNLB, CHST5, and GAL3ST2. In contrast, 2’-FL modulated the mRNA expression of the genes that express Golgi sulfotransferases genes. Furthermore, the combination of 2’-FL with Lac induced the mRNA expression of MUC2. This demonstrates that individual HMOS, such as 2’-FL, can have a direct and specific effect on the mRNA expression of genes involved in the maintenance of the mucus layer and the sulfonation of mucin secreted by goblet cells.
Many clinical trials have concluded that the use of 2’-FL enzymatically produced to be used in infant formula is safe, well tolerated and absorbed and excreted as human 2’-FL [29]. Although it requires more assessment, the direct modulation of the intestinal barrier function can be instrumental to treat disorders in which the integrity of the mucus layer or mucin is defective, such as helminth infections, cystic fibrosis, and inflammatory bowel disease [30].
The clinical studies studying the effects of 2’-FL in human milk substitutes are still limited, and the major setback is that while one or two HMOS are added to infant formula, human milk contains more than 200 structures [31]. Therefore, it remains unclear which other structures contained in HMOS can have a modulatory effect on goblet cells. Therefore, further studies on goblet and epithelial cells function, should include other individual HMOS. Studies on fucosylated and sialylated HMOS could also shed light on the structural traits that modulate epithelial responses.
Conclusions
Glycobiology is the field of study of the function and biology of glycans. Through the work presented in this thesis we are contributing to this field by bridging analytical techniques for the preparation or modification of dietary carbohydrates with functional studies. We evaluated the capacity of bLF, GOS and 2’-FL to affect the intestinal barrier function in
vitro. Specifically, we showed that the modification of the mannose and sialic acid content
on the chains on bLF glycans plays a role in the molecular basis for the immunomodulatory capacity mediated via TLRs. For the first time, we showed that the isolated glycans from bLF can affect the immune response in reporter monocytes and in human MoDCs. The identification of the effects of mannose and sialic acid on the glycans of bLF can improve the understanding of its contribution to health and disease and expand its applications. Additionally, our results indicate that the content of transgalactosylated oligosaccharides and the presence of β-glycosidic linkages in GOS affect differentially the mRNA expression of genes involved in the formation and maintenance of the intestinal mucus layer. Furthermore, we observed that lactose can have an additive effect on the overall impact of GOS on the function of goblet cells. This modulatory effect of goblet cell function was also displayed by the enzymatically produced FL and isolated HMOS. In addition, 2’-FL protected the barrier integrity by preventing changes in the TEER of an epithelial monolayer. Taken together, the results gathered in this thesis showed that diet-derived free and conjugated glycans target directly immunomodulatory and intestinal epithelial responses functions in vitro.
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Future perspectives
The concept of using food as medicine is not new. Hippocrates is credited with the quote “Let food be thy medicine and medicine be your food”. Nutrition is not only regarded as an intervention to improve health, it is recognized as a strategy to stabilise or reverse a pathological condition. Food components, such as glycans, are finding their way into the therapeutic field thanks to the acknowledgement that these molecules can have multiple targets and modulate biological responses in different ways.
The interest in the health benefits of glycans derived from diet is not only emerging faster thanks to the increased efforts to improve glycans characterisation or production methods, it has contributed to change the therapeutical paradigm “one disease, one target, one drug”. This paradigm dictates that one compound should have specific selectivity towards a molecule of interest for a set of symptoms. In consequence, this oversimplifies disease mechanisms. The understanding of the role of glycans in the prevention of pathogen adhesion or their contribution to the immune and epithelial intestinal responses has the potential to open venues to decode the mechanisms that reduce the chances to develop, for instance, viral or autoimmune diseases. Therefore, instead of developing one compound to fight a virus, the meaning of the interactions mediated by free and conjugated glycans in the host, provide the cues to identify how to deprive pathogens of the means to attach to host cells [32].
The understanding of how glycoconjugates or free glycans exert their functions and the complexity of their structures in the in vivo human situation is the challenge and future of glycobiology and, for instance, its application in infant nutrition. The first year of life is the most demanding stage for the growth and development of an infant. Compared to other stages inadequate nutrition during this period cannot be compensated by other foods. The composition of breast milk substitutes specially in terms of carbohydrate content is critical to shape the intestinal microbiota and stir the immune response for the infant health. The results from this thesis provide information about the effects of mannose, sialic acid, galactose, glucose, and fucose composition on dietary glycans in the intestinal epithelial response and the intestinal immune function. Despite that we attempted to reproduce temporal structural changes in lactation, this is a crucial aspect that requires extra attention in future research. A big challenge to the application of this knowledge to the infant nutrition sector is precisely the ability to reproduce the proper glycan composition that can fit the rapid changes of the developing infant physiology. For instance, the development of infant formulas for preterm infants. Low birth or pre-term infant are at risk to manifest lactose intolerance, which demands that infant formulas have low or no lactose content in order to prevent intestinal complications [33]. Preterm babies have big
caloric requirements that clash with a small gastric volume and a limited digestive capacity [34]. Additionally, the breast milk of the mother of a preterm infant differs from the milk produced by a mother whose child is born after 37 weeks [35]. This example illustrates how critical is the interpretation of the role of the structural composition of glycans during lactation in the different stages of infant growth.
Currently, the development of analytical techniques and microbiological engineering methods have opened venues for the isolation, characterization and production of specific glycans. Nonetheless, one limitation of the production of these functional carbohydrates is the capacity of manufacturers to produce these compounds in affordable large amounts that meet the increasing demands of consumers. Additionally, it is also relevant to optimize the methods required to purify the resulting mixtures while preserving their structure. By overcoming these challenges, the possibility to produce customized glycans can broad their application in the fields of clinical and non-clinical nutrition.
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