Human Milk Oligosaccharides: New Developments in the Management of Cow’s Milk Protein Allergy

Editor(s): Alexandra F. Santos, Lars Bode, Anna Nowak-Wegrzyn, Sophie Nutten.

Human Milk Oligosaccharides for Immune System Development
Professor Lars Bode

Breast milk is the gold standard in infant nutrition and plays a key role in infant survival and health. According to the editorial from The Lancet’s 2016 series on breastfeeding, “The deaths of 823,000 children and 20,000 mothers each year could be averted through universal breastfeeding, along with economic savings of US$300 billion.”1 Hence, research on the constituents of breast milk, as well as their mechanisms, could lead to better understanding of the role of breast milk in improving infant health. HMO are the third largest nonaqueous component of breast milk, after lactose and fat, and are present in concentrations of 5–15 g/L. Bovine milk, in comparison, contains approximately 100- to 1,000-fold lower concentration of oligosaccharides. HMO are complex sugars derived from combinations of five monosaccharides: glucose, galactose, N-acetylglucosamine, fucose, and sialic acid. The physiological functions of HMO depend on the final structure of the macromolecule resulting from the combination of these building blocks, as well as further chemical modifications, such as fucosylation or sialylation. Over the years, 150–200 distinct HMO have been identified. The  expression of individual HMO varies from mother to mother, and results of the Canadian CHILD study cohort of 1,200 mothers have revealed a large heterogeneity in the absolute concentration and relative abundance of HMO in breast milk, which is dependent on multiple intrinsic and extrinsic factors.2 Breast milk composition changes over the course of lactation in an individual mother, but intraindividual HMO levels are remarkably constant over short periods of time, i.e., within 1 week or within 1 day, although at the interindividual level, composition varies from mother to mother. HMO, once ingested, pass through the gut largely unchanged owing to their resistance to low pH and pancreatic and brush border enzymes. Approximately 1% are absorbed in the small intestine and appear in the urine, while 10–80% are excreted via faeces following bacterial degradation in the colon.3 HMO play numerous roles in the infant gut (Figure 1). They serve as prebiotics: a food source for beneficial bacteria residing in the gut.

In addition, HMO are protective against certain harmful pathogens owing to their antimicrobial properties and by preventing bacterial adhesion to the intestinal epithelium. HMO also have direct effects on the epithelial and immune cells, which may subsequently affect the gut microbial composition. Despite this growing body of research, there is still a vast amount of knowledge yet to be discovered regarding the role of HMO. Various approaches ranging from in vitro and ex vivo studies to in silico modelling to human cohort studies and clinical trials are being employed worldwide in research on HMO. The specific effects of individual HMO have already been demonstrated in in vitro and animal studies: the antimicrobial/antiadhesive effects of 2’-FL, lacto-N-tetraose, 3’-sialyllactose (3’-SL), and 6'-sialyllactose (6’-SL);4-6 the effects of 2’- FL, 3’-SL, 6’-SL, and LNnT on infant growth7-10 and neurodevelopment;11-14 and the effects of 2’- FL, 3’-SL, and 6’-SL on immune development in infants.15-19 Numerous human cohort studies have also revealed noteworthy associations between
HMO and infant health. Seppo et al.20 showed that infants who received breast milk with low levels of the HMO lacto-N-fucopentaose III (LNFP-III) were more likely to develop CMPA. The CHILD study has also shown that specific HMO profiles are associated with food sensitisation in infants.21 These findings are consistent with findings from a larger cohort who were followed for 18 years to investigate the association between HMO profiles of breast milk and development of allergies; it was not only individual HMO, but also groups and clusters of HMO that were predictive of allergic disease.22 Finally, Prof Bode cited two recent review articles by Triantis et al.23 and Doherty et al.,24 which summarised the benefits of HMO in immune-mediated disease and infections in infancy. In conclusion, HMO play a crucial role in infant health and current trends in HMO research hold the potential of alleviating immunologic conditions in infants who cannot be fed breast milk.

Benefits of Human Milk Oligosaccharides in the Management of Cow’s Milk Protein Allergy
Professor Anna Nowak-Wegrzyn

The gut microbiome in infants plays a key role in immune maturation and immunoregulation by modulating effector or tolerant responses to different antigens and balancing the activities of Th1 and Th2 immune cells.25 During infancy, the predominant beneficial bacteria in the gut are Bifidobacterium and lactobacilli;26 their metabolites, specifically short-chain fatty acids (SCFA), influence the development of the infant immune system through a variety of physiological processes. In breastfed
infants, breast milk influences gut microbiome diversity and promotes higher concentrations of Bifidobacterium and lactobacilli. In addition, certain constituents of breast milk (secretory IgA, HMO, lactoferrin, and antimicrobial peptides) help the infant’s immune system by acting as barriers to pathogenic bacteria in the gut or providing indirect immune support. Lactose in breast milk has been shown to upregulate defences, especially against gut infections, through antimicrobial peptides.27 Additionally, HMO act as substrates for Bifidobacterium, driving up levels of SCFA.

In comparison to infants without food allergies, the gut microbiome of infants with food allergies reportedly has lower levels of Bifidobacterium and lactobacilli, and higher levels of Clostridium, staphylococci, and Escherichia coli. When breastfeeding is not possible for any reason, extensively hydrolysed formula (EHF) with lactose should be used as the first-line choice in infants with CMPA. Lactose supplementation has been shown to result in an increase in bacterial counts for Bifidobacterium and lactobacilli
(p<0.01) and a concomitant decrease in the counts of harmful bacteria, such as Bacteroides and clostridia (p<0.05), in infants with CMPA.28 Moreover, the investigators reported an increase in concentrations of SCFA (p<0.03), especially acetate and butyrate, confirming the ability of lactose supplementation to restore a healthy gut microbiota. Of the SCFA, butyrate plays a prominent role in maintenance of intestinal epithelial and enterochromaffn cell integrity, as an energy source for colonocytes, mucin release, absorption of water, anti-inflammatory effects, interaction with T-regulatory (Treg) cells, and barrier function of the gastrointestinal tract.

Correspondingly, low faecal butyrate levels have been reported in allergic infants aged up to 1 year.29 Other factors known to be associated with infant allergies are low IgA levels, early antibiotic exposure, caesarean birth, microbial dysbiosis and immune disturbances, and impaired intestinal permeability. The effects of an atypical gut microbiome on predisposition to allergy commences as early as the neonatal stage. In an investigation of a birth cohort, it was reported that the group with the highest microbial diversity, including lower relative abundance of certain bacteria such as Bifidobacterium, Akkermansia, and Faecalibacterium and a higher concentration of specific IgE at 2 years, had the highest risk of developing allergies.30 Hence, neonatal gut microbiota influence susceptibility to childhood allergies via alterations in the gut
microenvironment. Lower respiratory tract infections (LRTI) are common in infants and young children aged 1–3 years and contribute significantly to morbidity and mortality,
healthcare resource use, and quality of life.31 Infants with CMPA have a higher propensity for developing infections, such as LRTI, with a subsequent higher use of antibiotics, which is itself a risk factor for allergies.32 Prof Nowak-Wegrzyn then focussed on the specific role of two HMO, 2’-FL and LNnT, in infants with CMPA. These neutral oligosaccharides are the most abundant HMO in breast milk, accounting for approximately 30% of the total HMO content.33-36 The individual concentrations
of these HMO is highest in the immediate post- partum period, which indicates a role in the development of the neonatal gut microbiota.33 Trials of infant formulas supplemented with 2’-FL and LNnT conducted in healthy infants showed that infants had a lower incidence of LRTI, bronchitis, and antibiotic usage in comparison with infants who received formula without HMO (relative risk for bronchitis: 0.37; 95% confidence interval [CI]: 0.18–0.75, relative risk for antibiotic use: 0.69; 95% CI: 0.51–0.92).37 These findings indicate that HMO are protective against dysbiosis. Another study investigating the hypoallergenicity of a whey-based EHF (w-EHF), supplemented with 1.0 g/L of 2’-FL and 0.5 g/L of LNnT (Althéra® HMO, Nestlé Health Science, Vevey, Switzerland), confirmed that >98% of the infants and young children aged <4 years with CMPA tolerated the supplemented w-EHF in both the modified intent-to-treat cohort and the per-protocol cohort.38 Thus, the w-EHF with additional 2’-FL and LNnT meets the clinical criteria for hypoallergenicity set by the American Academy of Pediatrics (AAP) and can be recommended for the management of CMPA in infants and young children. Results from the international, multicentre CINNAMON trial investigating the effect of the w-EHF supplemented with 1.0 g/L of 2’-FL and 0.5 g/L of LNnT with a lowered protein content, on infants with CMPA showed that there was no difference in growth using weight-for-age and length-for-age z-scores at 4 months post- baseline between supplemented and control formula.39 An interesting finding from this trial was the observed reduction in relative risk for infections (LRTI, upper RTI, otitis media, and gastrointestinal tract infection), as well as related medication (antibiotics and antipyretics) usage. Moreover, there was a reduction in the mean number of infectious episodes for LRTI (p= not significant) and upper RTI (p=0.003) in infants receiving the w-EHF supplemented with 2’-FL and LNnT. Prof Nowak-Wegrzyn also shared further secondary data from the CINNAMON trial (presented at FAAM-EUROBAT Digital 2020) that showed, for the first time, the existence of a lower α-diversity in the gut microbiota of infants who received the w-EHF supplemented with 2’- FL and LNnT (Figure 2).40 Although it is assumed that diversity is beneficial for the development of infant gut microbiota, healthy breastfed infants are found to have lower diversity, with gut microbiota dominated by bifidobacteria and lactobacilli. Pedersen et al.40 reported that microbial richness and Shannon index (a measure of microbiome diversity) were noticeably lower in infants with CMPA who had received the w-EHF supplemented with 2’-FL and LNnT, which reached a statistical significance by 12 months of age (p<0.001).


 
Moreover, the shift of microbial clusters from ‘early type’ to ‘late type’ gut microbiome, which is typically seen in infants with CMPA around 1 year of age, appeared to have been restored to the early type with more beneficial bacteria, such as Actinobacteria, in infants fed the w-EHF supplemented with 2’-FL and LNnT (p=0.014). Prof Nowak-Wegrzyn concluded by emphasising the role played by HMO in rectifying the dysregulation of gut microbiome in infants predisposed to allergic disease. w-EHF containing 2’-FL and LNnT (Althéra HMO) has been shown to be safe and well tolerated in infants with CMPA and appears to be protective against several infections.

Moreover, w-EHF supplemented with 2’-FL and LNnT appeared to delay the premature shift in gut microbiota to the adult type, a phenomenon indicative of future allergy development in infants who receive little or no breastfeeding.

Impact of Human Milk Oligosaccharides in Food Allergy
Doctor Sophie Nutten

Although breast milk is recommended for prevention of allergic diseases in high-risk
infants, the exact underlying mechanisms are yet to be completely elucidated. Breastfeeding has been inconsistently associated with allergic conditions,41 reflecting the differences in breast milk constituents in different settings and populations. Breast milk contains various factors that affect immune development in the neonatal gut and could have an impact on allergic diseases. HMO are one of the most extensively studied major bioactive milk components. While devoid of a particularly nutritive value, HMO have numerous functions, including maintenance of the infant gut microbiome and development of infant immune system. Given these findings, it is suggested that HMO could also have a protective role in the development of allergies. Dr Nutten shared data from recent observational studies. A Finnish observational study that investigated the role of fucosyltransferase 2 (FUT2)-dependent HMO in allergic disease later in life showed a lower incidence of IgE-associated allergies and eczema at age 2 years in infants delivered by caesarean section who were fed breast milk containing FUT2-dependent HMO (p<0.1).35 The expression of 2’-FL, one of the HMO regulated by the FUT2 gene, varies substantially in mothers with FUT2 polymorphism and hence the investigators probed further for an association between 2’-FL expression and allergic manifestation. Of note, the incidence of IgE-associated allergies and eczema in the caesarean-born infants was inversely proportional to the level of 2’-FL in the breast
milk received during breastfeeding. At 2 years, a lower incidence of IgE-associated allergies and eczema was seen in caesarean-born infants fed breast milk containing FUT2-dependent HMO (Figure 3).

In the aforementioned study by Seppo et al.,20 besides LNFP-III, the investigators also
noted substantially lower levels of other HMO (disialyllacto-N-tetraose, 6’SL, and LNFP-I) in the breast milk of mothers whose infants were at a higher risk of CMPA. Despite these findings, it is unclear whether individual HMO are always implicated in the development of allergies. An analysis of a Canadian cohort that investigated the role of 19 different HMO could not identify any association between these individual HMO and food sensitisation.21 However, the investigators noticed a difference in pattern of HMO composition between infants with and without food sensitisation: infants who received
breast milk with higher levels of six HMO and lower levels of four HMO were associated with food sensitisation in the first year of life. Another recently published report on a long- term Australian study in a high allergy risk birth cohort found that exposure to acidic Lewis HMO profiles was associated with a higher risk of allergic disease and asthma (odds ratio: 5.82; 95% CI: 1.59–21.23), while exposure to breast milk with an acidic-predominant HMO profile was associated with reduced food sensitisation (odds
ratio: 0.08; 95% CI: 0.01–0.67).22 This leads to the conclusion that it is not only levels of individual HMO but also the overall composition profile of HMO that influence development of allergies. Dr Nutten also shared unpublished data from a German cohort in which the investigators observed a nonlinear association between specific levels of 2’-FL and LNnT and incidence of infant allergy.


Certain FUT3 polymorphisms are known to result in increased levels of 2’-FL and this could perhaps explain the nonlinear association; however, further studies are required to establish causality. Having established a case for the role of HMO composition in the development of food allergies and its implication on infant nutrition, Dr Nutten proceeded to discuss the results of preclinical studies that investigated the role of HMO in animal models of allergies. In an ovalbumin-sensitised mouse model of food allergy, supplementation with the HMO 2’-FL or 6’-SL could decrease the symptoms of food allergy, measured by diarrhoea score, in mice.42 Furthermore, the investigators showed an increase in Treg cells, as well as an indirect stabilisation of mast cells, which are largely responsible for allergic response in the mouse intestine.

This observation established mechanisms through which HMO supplementation resulted in the decrease of the treated animal’s symptoms. In another recent study, supplementation with 3’-SL in two different mouse models of atopic dermatitis was shown to result in the protection against allergic sensitisation (decrease in IgE production in a dose-dependent manner) and symptoms associated with a decrease in mast cell numbers.43 These data highlight the effectiveness of HMO in modulating the immune response at the whole animal level. Finally, Dr Nutten shared unpublished data from in vitro mechanistic studies on 2’-FL and LNnT against food allergy. In a cellular model using peripheral blood mononuclear cells, treatment with a mixture of 2’-FL and LNnT resulted in a decrease in the production of IL-5, one of the key modulators of allergic disease. Moreover, incubation of mast cells with the 2’-FL–LNnT mixture resulted in a decreased degranulation of the former, indicating that 2’-FL/LNnT stabilised mast cells at tested concentrations. To investigate the effects of 2’-FL and LNnT on the gut epithelial barrier, the investigators preincubated intestinal epithelial cell lines, CaCo-2 and HT29- MTX treated with TNFα and IFNγ to mimic inflammatory conditions, with 2’-FL and LNnT. Pretreated cellular monolayers were found to have a significantly lower permeability, measured by the FITC-dextran-4 flux, in comparison with untreated cells. Progressing to animal studies, in the ovalbumin-choleratoxin mucosal sensitisation mouse model, supplementation of the animals’ diet with 2’-FL and LNnT at different concentrations reduced sensitisation and increased Treg counts in the animals. Moreover, a decrease in mast cell numbers and mast cell
degranulation was observed in animals who received supplementation. Similar results were noticed in the Aspergillosis fumigatus-induced epicutaneous sensitisation mouse model; animals with diets supplemented with 2’-FL and LNnT displayed reduced sensitisation compared with control animals. Moreover, the investigators noted a dose-dependent effect of supplementation on gut microbiota composition, the highest difference reaching up to 10%. In conclusion, both human and preclinical data are promising. Yet, further investigation on the potential of HMO supplementation for allergy management in infancy is warranted.

References
1. Breastfeeding: achieving the new normal. Lancet. 2016;387(10017):404.
2. Azad MA et al. Human milk oligosaccharide concentrations are associated with multiple fixed and modifiable maternal characteristics, environmental factors, and feeding practices. J Nutr. 2018;148(11):1733-42.
3. Bode L. Human milk oligosaccharides: every baby needs a sugar mama. Glycobiology. 2012;22(9):1147-62.
4. Lin AE et al. Human milk oligosaccharides inhibit growth of group B Streptococcus. J Biol Chem. 2017;292(27):11243-9.
5. Jantscher-Krenn E et al. Human milk oligosaccharides reduced Entamoeba histolytica attachment and cytotoxicity in vitro. Br J Nutr. 2012;108(10):1839-46.
6. Laucirica DR et al. Milk oligosaccharides inhibit human rotavirus infectivity in MA104 cells. J Nutr. 2017;147(9):1709-14.
7. Alderete TL et al. Associations between human milk oligosaccharides
and infant body composition in the first 6 mo of life. Am J Clin Nutr. 2015;102(6):1381-8.
8. Larsson MW et al. Human milk oligosaccharide composition is associated with excessive weight gain during exclusive breastfeeding– an explorative study. Front Pediatr. 2019;7:297.
9. Lagström H et al. Associations between human milk oligosaccharides and growth in
infancy and early childhood. Am J Clin Nutr. 2020;111(4):769-78.
10. Charbonneau MR et al. Sialylated milk oligosaccharides promote microbiota-dependent growth in models of infant undernutrition. Cell. 2016;164(5):859-71.
11. Vázquez E et al. Effects of a human milk oligosaccharide, 2’-fucosyllactose, on hippocampal long-term potentiation and learning capabilities in rodents. J Nutr Biochem. 2015;26(5):455-65.
12. Oliveros E et al. Oral supplementation of 2‘-fucosyllactose during lactation improves memory and learning in rats. J Nutr Biochem. 2016;31:20-7.
13. Berger PK et al. Human milk oligosaccharide 2’-fucosyllactose links feedings at 1 month to cognitive development at 24 months in infants of normal and overweight mothers. PLoS One. 2020;15(2):e0228323.
14. Wang B. Molecular mechanism underlying sialic acid as an essential nutrient for brain development and cognition. Adv Nutr. 2012;3(3):465S-72S.
15. Newburg DS et al. Lactodifucotetrasose, a human milk oligosaccharide, attenuates platelet function and inflammatory cytokine release. J Throm Thrombolysis. 2016;42(1):46-55.
16. Bode L et al. Human milk oligosaccharides reduce platelet- neutrophil complex formation leading to a decrease in neutrophil β2 integrin expression. J Leukoc Biol. 2004;76(4):820-6.
17. Bode L et al. Inhibition of monocyte, lymphocyte, and neutrophil adhesion to endothelial cells by human milk oligosaccharides. Thromb Haemost. 2004;92(6):1402-10.
18. Eiwegger T et al. Human milk-derived oligosaccharides and plant-derived
oligosaccharides stimulate cytokine production of cord blood T-cells in vitro. Pediatr Res. 2004;56(4):536- 40.
19. Cornstock SS et al. Select human milk oligosaccharides directly modulate
peripheral blood mononuclear cells isolated from 10-d-old pigs. Br J Nutr. 2014;111(5):819-28.
20. Seppo AE et al. Human milk oligosaccharides and development of
cow’s milk allergy in infants. J Allergy Clin Immunol. 2017;139(2):708-11.
21. Miliku K et al. Human milk oligosaccharide profiles and food sensitization among infants in the CHILD study. Allergy. 2018;73(10):2070-3.
22. Lodge CJ et al. Human milk oligosaccharide profiles and allergic disease up to 18 years. J Allergy Clin Immunol. 2020;S0091- 6749(20):30956-8.
23. Triantis V et al. Immunological effects of human milk oligosaccharides. Front Pediatr. 2018;6:190.
24. Doherty AM et al. Human milk oligosaccharides and associations with immune-mediated disease and infection in childhood: a systematic review. Front Pediatr. 2018;6:91.
25. Pascal M et al. Microbiome and allergic diseases. Front Immunol. 2018;9:1584.
26. Tanaka M, Nakayama J. Development of the gut microbiota in infancy and its impact on health in later life. Allergol Int. 2017;66(4):515-22.
27. Cederlund A et al. Lactose in human breast milk an inducer of innate immunity with implications for a role in intestinal homeostasis. PLoS One. 2013;8(1):e53876.
28. Francavilla R et al. Effects of lactose on gut microbiota and metabolome of infants with cow’s milk allergy. Pediatr Allergy Immunol. 2012;23(5):420-7.
29. Roduit C et al. High levels of butyrate and proprionate in early life are associated with protection against atopy. Allergy. 2019;74(4):799-809.
30. Fujimura KE et al. Neonatal gut microbiata associates with childhood multi sensitized atopy and T cell differentiation. Nat Med. 2016;22(10):1187-91.
31. Dainelli L et al. Health and economic burden of lower respiratory tract
infection in children under 3 years with and without cow's milk protein allergy. Abstract. EAACI PAAM, 17-19 October, 2019.
32. Woicka-Koljwa K et al. Food allergy is associated with recurrent respiratory tract infections during childhood. Postepy Dermatol Allergol. 2016;33(2):109-13.
33. Austin S et al. Temporal change of the content of 10 oligosaccharides in the milk of Chinese urban mothers. Nutrients. 2016;8(6):346.
34. Austin S et al. Human milk oligosaccharides in the milk of mothers delivering term versus preterm infants. Nutrients. 2019;11(6):1282.
35. Sprenger N et al. Longitudinal change of selected human milk oligosaccharides and association to infants’ growth, an observatory, single center, longitudinal cohort study.
PLoS One. 2017;12(2):e0171814.
36. Samuel TM et al. Impact of maternal characteristics on human milk oligosaccharide composition over the first 4 months of lactation in a cohort of health European mothers. Sci Rep. 2019;9(1):11767.
37. Puccio G et al. Effects of infant formula with human milk oligosaccharides on growth and morbidity: a randomized multicenter trial. J Pediatr Gastroenterol Nutr. 2017;64(4):624-31.
38. Nowak-Wegrzyn A et al. Confirmed hypoallergenicity of a novel whey- based extensively hydrolyzed infant formula containing two human milk oligosaccharides. Nutrients. 2019;11(7):1447.
39. Vandenplas Y et al. Growth tolerance and safety of an extensively hydrolyzed formula containing two human milk oligosaccharides in infants with cow’s milk protein allergy. Oral abstract presentation. EAACI PAAM, 17-19 October, 2019.
40. Pedersen H et al. An extensively hydrolysed formula supplemented with two human milk oligosaccharides (HMO) shapes the gut microbiome in infants with cow’s milk protein allergy (CMPA). Abstract FP51. FAAM–EUROBAT Digital 2020, 16-17 October, 2020
41. Lodge CJ et al. Breastfeeding and asthma and allergies: a systematic review and meta-analysis. Acta Paediatr. 2015;104(S467):38-53.
42. Castillo-Courtade L et al. Attenuation of food allergy symptoms following treatment with human milk oligosaccharides in a mouse model. Allergy. 2015;70(9):1091-102.
43. Kang LJ et al. 3’-sialyllactose prebiotics prevent skin inflammation via regulatory T cell differentiation in atopic dermatitis mouse models. Sci Rep. 2020;10(1):5603.