First, Angeloni et al. demonstrated that sialylated HMO fractions (especially 3’SL) induce a modification of the extracellular glycosylation pattern of the Caco-2 cell line (37). This modification was associated with a 50% reduction in enteropathogenic E. coli in Caco-2 cells treated with 3’-SL compared to untreated cells ( Figure 3 ). Moreover, Chunli Kong et al. demonstrated that 3’-FL increased albumin absorption and the presence of heparan sulfate (HS) and hyaluronic acid (HA) in the glycocalyx of a Caco-2 cell line (38).

In 2008, Kuntz et al. observed that neutral and acidic fractions of HMOs induced cell growth inhibition of small intestinal epithelial cells (HIECs), HT-29 cells and Caco-2 cells in vitro (39). Furthermore, these two fractions of HMOs induced HT-29 and HIEC differentiation but not Caco-2 cell differentiation. Finally, only neutral HMOs induced HT-29 and HIEC apoptosis characterized by caspase 3 cleavage but not Caco-2 cells supporting the hypothesis of differentiation-associated processes. They concluded that HMO are able to induce growth inhibition in intestinal cells through two different mechanisms, by suppressing cell cycle progression through induction of differentiation and/or by influencing apoptosis. By inducing the differentiation of intestinal cells in vitro, HMOs may influence the maturation of the intestinal barrier ( Figure 3 ). Indeed, Holscher et al. determined that neutral HMOs induced the differentiation of HT-29 cells in villus enterocyte-like cells, promoting digestive capacities of the epithelial barrier (40). Finally, Perdijk and colleagues confirmed that sialylated HMOs influence intestinal cell homeostasis in vitro, highlighting the beneficial effect of HMOs on epithelial cells (41).

In conclusion, some studies demonstrated that HMOs are able to modify the extracellular glycosylation of epithelial cells limiting the adhesion of some pathogens and enhancing albumin absorption. A couple of another studies agree on the effects of HMO on the modulation of the cell cycle preventing cell growth and inducing cell differentiation in villus enterocyte-like cells leading to a potential better absorption of nutrients. As the development and maturation of digestive and absorptive processes depend on differentiation, studies show that HMOs are effective at influencing various stages in gastrointestinal development.

HMOs can also protect the intestine by inducing the production of mucin ( Figure 3 ). Mucin is the main protein that composes intestinal mucus and protects against intestinal infection (42). Indeed, Wu et al. demonstrated that exposure of human intestinal epithelial cells and intestinal organoids to HMOs induced expression of Muc2, a chaperone protein, that then activated disulfide isomerase (PDI) in vitro (43). They also showed an increase in Muc2 expression and a decrease in intestinal permeability in an HMO-supplemented murine model.

Suligoj et al. demonstrated in their study that 2’-FL alone or 2’-FL mixed with LNnT induced the expression of the claudin-8 gene in Caco-2 cell monolayers and that the fermentation of 2’-FL induced the expression of claudin-5 in gut-on-chips from human organoids derived from proximal, transverse, and distal colon biopsies (44). Claudin proteins play a crucial role in tight junctions between two epithelial cells, blocking the passage of molecules in the paracellular pathway and thus decreasing the permeability of the gut (45) ( Figure 3 ).HMOs incubated with B. longum infantis increased mRNA transcription of ZO-1 and the junctional adhesion molecule (JAM-A) in Caco-2 cells and HT-29 cells (46). Furthermore, the authors demonstrated that HMOs prevent the internalization of occludins inside Caco-2 and HT-29 cell lines (46). However, Sua Kim et al. demonstrated that ZO-1 and occludins are necessary to maintain transepithelial electrical resistance and thus to preserve the function of the proximal tubular epithelium (47). ZO-1 (and ZO-2) are involved in the regulation of tight junction assembly, and JAM-A proteins regulate the structural organization of tight junctions, which is essential for the impermeability of the epithelial barrier (48).

In conclusion, these studies highlight the efficiency of HMOs to protect and strengthen the impermeability of intestinal barrier by modulating mRNA transcription and protein expression of several key molecules such as ZO-1, muc2 and claudin.

HMOs can affect the protein expression of epithelial cells because a small portion of HMOs are absorbed by intestinal cells ( Figure 3 ). Acidic oligosaccharides were shown to only pass through intestinal cells via the nonspecific paracellular route, and neutral oligosaccharides were shown to pass through intestinal cells via the paracellular or transcellular route involving the receptor pathway (49). The action of HMOs on gene expression was also demonstrated in vivo, showing that supplementation with sialylated HMOs in rats affects intestinal gene expression and modulates the intestinal glycome (50).Sialylated HMOs reduce the secretion of IL-12 in Caco-2 cells and decrease the gene expression of IL-12, IL-8, and TNFα (51). NF-κB gene expression and protein translocation were also reduced by HMOs in that study. These effects are mediated by the induction of the nuclear receptor PPARγ, leading to the regulation of anti-inflammatory peptidoglycan recognition protein 3 activation. It was also shown in HT-29 and Hep-2 cell lines that 2’-FL induced a decrease of 80% in C. jejuni invasion, and this decrease was associated with suppression of mucosal proinflammatory cytokine secretion (IL-8, IL-1β and neutrophil chemoattractant macrophage inflammatory protein 2) (52).

One in vitro study revealed that 2’-FL could have promising beneficial effects on allergy signaling pathways by selectively inhibiting IL-8 and CCL20 release by T-84 and HT-29 in response to the antigen-antibody complex in the PPARγ-dependent pathway (53) ( Figure 3 ).

In conclusion, all these studies converge to demonstrate that HMOs reduce pro-inflammatory cytokines secretion by epithelial cells.

Zhuo-Teng Yu et al. revealed that 2’-FL and 3’-FL induced Bifidobacteria and Bacteroides growth in vitro. Conversely, these 2 HMOs do not generate major growth effects in Lactobacillus, Enterococcus, and Streptococcus (57). Additionally, 3’SL and 6’SL induced moderate growth of Bifidobacteria and Bacteroides (57), two commensal bacteria known to be good for human health (58–60). Conversely, Enterococcus (61) and Streptococcus (62) are two bacteria that induce human infection. Other in vitro studies were conducted and demonstrated that HMOs are able to decrease the colonization (63) and growth (64) of group B Streptococcus. Moreover, Salli and colleagues demonstrated that 2’-FL induces an increase in members of the phyla Firmicutes and Actinobacteria in vitro (65); Bifidobacteria that are commonly found in the gut belong to the phylum Actinobacteria (66). In this study, 2’-FL induced a decrease in members of the Proteobacteria phylum that known to cause different diseases (67). Finally, Hoeflinger et al. demonstrated that no Enterobacteriaceae strains grew in the presence of 2’-FL, 6’SL or LNnT (68). In conclusion, all in vitro studies agree that HMOs not only induce the emergence and proliferation of bacteria that benefit health but also decrease the frequency of harmful bacteria.

Interestingly, a very recent study demonstrated that HMOs such as 2’-FL and LNT are hydrolyzed by Akkermansia muciniphila (69). This bacterium is known to colonize the mucus layer of the human intestinal tract and is associated with decreases in obesity, type 2 diabetes, inflammatory bowel disease, and appendicitis (69).

To summarize, these in vitro studies highlight the ability of HMOs to modulate the microbiota inducing on the one hand the growth of bacteria strains benefic for the health and on the other hand the decrease of bacteria strains involved in bacterial infections.

Two in vivo studies were conducted in newborn pigs supplemented with 2’-FL (10 g/L for 7 days), and both revealed a trend towards an increase in the Lachnospiraceae genus and a decrease in Enterobacteria in the intestine, inducing a nonsignificant diminution of diarrhea in newborn pigs (70, 71) ( Figure 4 ).

In clinical trials in humans, HMOs indicated their ability to modulate the microbiota of breastfed preterm infants and breastfed caesarian-born infants, altering the microbiota to be more similar to that of breastfed to term vaginally born infants (increasing Firmicutes and Bifidobacteria and decreasing Enterococcus) (72, 73). Interestingly, Bifidobacteria and Bacteroides were found in the infant fecal microbiota of one-month-old breastfed children only if their mother secreted 2’-FL and LNFP in milk (74). This finding was correlated with limited infection (75) and protection against obesity (76). HMOs also have an influence on the colon microbiota and are directly fermented by bacteria from the colon (74) ( Figure 4 ). Indeed, the composition of the microbiota is different depending on the HMOs consumed (74). For example, the consumption of all HMOs (nonfucosylated neutral, fucosylated, and sialylated) by one-month-old children induces a higher abundance of Bifidobacteria than the consumption of only one type of HMO (74). Furthermore, Wang et al. highlighted the correlation between HMO consumption and fecal microbiota composition (77). Indeed, 2’-FL supplementation was positively correlated with the abundance of Bacteroides and negatively correlated with Veillonella, Enterococcus, and Rothia in the feces of infants at 3 months postpartum.

As previously mentioned, the mother secreting status influences the HMO composition in breast milk. Therefore, the presence or absence of HMOs modulates the infant microbiota (78). It was shown that infants fed by secreting mothers had a higher relative abundance of Bifidobacterium strains and Bacteroides strains in feces than infants fed by nonsecretor mothers. Additionally, infants fed by secreting mothers presented fewer Enterobacteria, Clostridia, and Streptococci than infants fed by nonsecretor mothers. Another clinical trial demonstrated that Prevotella was not detected in stools from secretor mothers and their children (79). Similar to previous studies, Bifidobacterium abundance was increased in the stools of children from secretor mothers compared to children from nonsecretor mothers, and this effect was still relevant at 2 to 3 years of age.

Finally, it was shown that the consumption of acidic HMOs protects against microbial structure changes due to stress (80), and neutral HMOs are a selective substrate for some strains of Bifidobacteria (81).

In conclusion, clinical trials demonstrate the importance of the secretor status of mothers for the presence of HMOs in breastmilk and in the composition of the infant microbiota. These clinical trials correlate with the beneficial effects of HMO postnatal consumption and diverse and rich microbiota compositions that are beneficial for health.

HMOs can also modify the fermentation activity of the gut microbiota, especially short-chain fatty acid (SCFA) production ( Figure 4 ). SCFAs are an important source of energy for enterocytes and are key signaling molecules for the maintenance of gut health. HMOs can indirectly increase the production of SCFAs, and these augmented levels are mediated by Bifidobacteria species such as B. infantis (82), B. bifidum (83) and B. breve (84). Other bacteria able to ferment HMOs such as Bacteroidetes, produce abundant lactate and other SCFAs and consumption of 3’SL and 6’SL by Bifidobacterium and Bacteroides induces greater neuraminidase activity (57). High neuraminidases activity was nevertheless, shown to be involved in human disorders such as neurodegenerative disorders, cancers, and infectious diseases (85). Furthermore, HMOs increase butyrate production in the colon thanks to their fermentation activities (44) ( Figure 4 ).

Human trials and experimental murine models have wildly shown that increasing SCFAs through a high-fiber diet or direct supplementation have beneficial health effects. However, the effects of too high SCFAs concentration on the host has to be investigated, has one study found that higher SCFA concentrations were associated with obesity and hypertension in the adult (86).

Because HMOs mediate changes in the composition of the infant microbiota or in the intestinal epithelial cell response, they may indirectly affect the infant immune system. Many in vitro studies suggest that HMOs can also directly modulate immune responses ( Figure 5 ). Indeed, HMOs may act either locally, on cells of mucosa-associated lymphoid tissues, or at a systemic level, as 1-5% of HMOs are absorbed and reach the circulatory system (89).

It was first necessary to confirm that HMOs were well tolerated and had no side effects (118) ( Table 1 ).

Several studies conducted on newborns fed HMO-supplemented formulas demonstrated digestive tolerance of HMOs and identical child growth (weight, length, and head circumference) compared to breastfed infants (12–14, 117) ( Table 1 ). HMOs induce better sleep and a less important pathogens colonization leading to less colic and more Bifidobacterium present in the colon (14). Sprenger et al. (9) demonstrated that children breastfed for 4 months by secreting mothers (with a secretion of 2’-FL) had no difference in weight, size, or body mass index compared to breastfed children from nonsecretor mothers ( Table 1 ). No difference in cranial box circumference was described (119). Finally, it was also shown in pigs that supplementation with 2’-FL improved cognitive performance and influenced brain development (120). All these studies highlight that HMOs do not induce side effects on newborns breastfed by secretor mother or fed with a HMOs-supplemented formula. Nevertheless, it would be interesting to follow these children up to a certain age to see if differences appear in the long term or not.

Because HMOs do not present side effects, they were tested to determine whether they induce a reduction in babies’ ailments. Supplemented formula-fed infants who were cesarean born had softer stools and a lower incidence of colic at 4 months of life than nonsupplemented children (14) ( Table 1 ). They had a shorter period of bronchitis at 4, 6, and 12 months, fewer respiratory tract infections and a reduction in antibiotic and antipyretic uses compared to nonsupplemented children (14). It would have been interesting in this study to compare the results obtained in the supplemented formula-fed group with babies fed by secretor mothers to compare the effects of natural HMOs intake versus supplemented formula intake. HMOs are also associated with a reduction in diarrhea induced by Campylobacter during the first 2 years of life of children fed breast milk with a high concentration of 2’-FL compared to those fed with a low level of 2’-FL in the milk highlighting also the importance of the concentration of HMOs in the breast milk (11) ( Table 1 ). In parallel, an important study revealed a decrease in mortality in noninfected children born to HIV-infected mothers due to HMOs in breast milk compared to noninfected children who were not breastfed (11).

Several studies highlighted the protective effects of HMOs against infections. HMOs act as soluble receptors of bacteria or modulate target cells, inhibiting the pathogen-host cell interaction. Indeed, Angeloni et al. demonstrated that 3’SL exposure induces a change in the glycocalyx composition of Caco-2 cells (37). The expression of α2-3- and α2-6-linked sialic acid residues was downregulated on the cell surface, leading to a decrease in pathogen receptors on the host cell surface and thus a decrease in bacterial binding. A decrease in virus binding to DLD1 intestinal cells by HMOs was also highlighted (121). Interestingly, this particular effect of HMOs is not limited to intestinal cells. Indeed, Andersson et al. demonstrated that human milk inhibits the attachment of bacteria and viruses on pharyngeal and buccal epithelial cells (122). It was demonstrated that the anti-adhesive effect was due to the low molecular weight fraction of human milk, particularly to the oligosaccharides present in this fraction.

Furthermore, HMOs act as antimicrobial agents due to their ability to bind pathogens such as Streptococcus strains directly in the gut (123). In an in vitro study conducted by Weichert et al., the use of X-ray crystallography demonstrated that 2’-FL and 3’-FL are able to bind norovirus, linking in the HGBA pocket of the virus, and inhibiting its cytotoxic activity (124). They also demonstrated that HMOs can be direct substrates for pathogens, preventing infection of host cells. Finally, Lin et al. highlighted that sialic acid HMOs protect epithelial cells by preventing the internalization of Escherichia coli in UPEC cells in vitro, inhibiting the infection of the cells (125).

Finally, the anti-adhesive properties of HMOs also applied to some parasitic protozoa. For example, Entamoeba histolytica, (a protozoa that destroys the epithelium of the large intestine and can occur in the liver, lungs or spleen) requires for the infection to attach to the host’s colon mucosa. Parasites that cannot attach are excreted in faeces and do not cause disease. It was shown that some HMOs significantly reduce the binding and cytotoxicity of E. histolytica during in vivo assays (118). This may explain why breastfed infants are less likely to be infected with E. histolytica or other protozoa than formula-fed infants. To summarize, HMOs can prevent infection by 1) modulating virus receptor expression on the surface of the cell, 2) directly binding pathogens inhibiting their adhesion, and 3) preventing internalization through the gut.

Finally, it was shown in animal models that HMOs had protective effects on several non-communicable diseases. 3’SL supplementation is able to prevent the development of obesity in mice and to prevent type 2 diabetes and cardiovascular diseases (126). In the same way, HMOs in breastmilk seem to protect children against obesity until 4 years of age compared to children who were never breastfed (119).

Allergies are a major health issue in developed countries, and there is currently no effective therapeutic strategy to cope with allergies. Allergy pathologies are associated with a breakdown of immune tolerance (127), microbiota dysbiosis (128) and an increase in epithelial permeability (129). They may be observed within the first month of life, suggesting the possibility of early childhood preprogramming these biological systems and possibly reducing the risk of allergy development. Due to their beneficial effects on the microbiota, gut epithelial barrier and immune system, HMOs have been tested to treat or prevent allergic diseases. Today, only some preclinical and clinical studies have been conducted in this context.

In a mouse model of food allergies, HMOs were shown to be good candidates to treat or prevent allergic symptoms. Indeed, allergic mice supplemented with 2’-FL had a decrease in allergic symptoms such as diarrhea and hypothermia and a suppression of protease 1 secretion from mast cells mediated by antigen compared to non-supplemented allergic mice (130). The authors also demonstrated a decrease in intestinal mast cell frequency and an increase in CD4⁺CD25⁺IL10⁺ regulatory T cells in mesenteric lymph nodes and Peyer’s patches. Furthermore, they observed a reduction in the passive response to skin anaphylaxis. Recently, Liu et al., working on a β-lactoglobulin-induced milk allergic mouse model, demonstrated that supplementation with 2’-FL for 4 weeks decreased IgE and β-lactoglobulin-specific IgE in the serum and increased the levels of the anti-inflammatory cytokines IL-10, TGF-β, and IFNγ compared to allergic mice untreated with 2’-FL, thus preventing allergy development (131).

These encouraging data from preclinical models need to be confirmed by other studies in other allergic contexts, such as asthma or atopic dermatitis, and the mechanistic effects of HMOs must be deciphered.

Most human studies have correlated the presence of HMOs in breastmilk and the development of allergies in infants, but the results are controversial.

On the one hand, Seppo et al. demonstrated that a high level of LNFPIII in breastmilk protected infants against cow’s milk allergy compared to children fed breastmilk containing a low level of LNFPIII (16). On the other hand, Miliku et al. showed that HMO composition in human milk is associated with food sensitization in the first year of life, which can later lead to food allergies (18). Lodge et al. demonstrated that compared with children exposed to the neutral Lewis HMO profile, exposure to acidic Lewis HMOs was associated with a higher risk of allergic disease and asthma during childhood, whereas exposure to the acidic-predominant profile was associated with a reduced risk of food sensitization (20). Finally, the observational clinical trial conducted by Sprenger et al. (132) revealed that infants born by cesarean section and having a high hereditary risk for allergies might have a lower risk of manifesting IgE-associated eczema at 2 years, but not at 5 years of age, when fed breast milk with FUT2-dependent milk oligosaccharides. Further studies with larger cohorts and especially randomized controlled intervention trials are required to build on these preliminary observations.

In an interventional study, Nowak-Wegrzyn et al. attempted to determine whether an extensively hydrolyzed formula supplemented with two HMOs was tolerated by infants allergic to cow’s milk (19). They demonstrated that the whey-based extensively hydrolyzed formula supplemented with 2’-FL and LNnT met the clinical hypoallergenic criteria and could be recommended for the management of cow’s milk protein allergy in infants and young children.

These different results obtained in clinical trials are contradictory because the studies were conducted in different ways (time of supplementation, concentration, type of HMOs), complicating the understanding of HMO effects on allergic disease. New strategies could be explored to clarify the HMO effect.