From homeostasis to dysregulation: human milk, microbial metabolites, and the preterm intestinal microenvironment in necrotizing enterocolitis

Introduction

The gastrointestinal tract, the body’s largest immunological organ, maintains a delicate equilibrium: tolerating nutrients and commensal microbes while limiting access of pathogens. This equilibrium relies on a well-functioning mucosal barrier, which comprises intestinal epithelial cells (IECs), a mucus layer, and immune cells. While premature delivery disrupts normal development of the above system: truncated in-utero development results in hyperpermeable tight junctions, deficient Paneth cell-mediated antimicrobial defense, impaired macrophage/T-cell function, and dysregulated Toll-like receptor 4 (TLR4) signaling (1,2). These vulnerabilities, compounded by early enteral feeding and microbial colonization, may create a storm for necrotizing enterocolitis (NEC)—a devastating disease predominating in preterm infants, with mortality exceeding 30% in surgical cases (3).

Prematurity, microbiota dysbiosis, and the absence of human milk feeding are established NEC risk factors (4), and interventions targeting these areas remain inconclusive. Clinical studies on early antibiotic exposure in preterm infants show conflicting results regarding its impact on NEC risk, possibly indicating a time-dependent cumulative effect and raising safety concerns related to antibiotic overuse (5,6). In addition, although probiotic supplements have demonstrated some promising findings, significant variations exist in the probiotic strains and dosages used across different studies, and the optimal dosage and strain combinations remain to be established (6). Pharmacologic approaches, including TLR4 inhibitors and melatonin, have not achieved translation (4,7). Critically, human breast milk remains as the most significant and consistently protective factor against NEC, reducing incidence by up to 30% compared to formula (8). Although the microbial composition of human milk differs significantly from that of infant formula (9), a recent study indicates that the protective effect of human milk against NEC is not mediated by its microbiota (10).Therefore, bioactive nutrients in human milk and changes in the infant’s gut microbiota/metabolites after feeding may represent critical intervention targets for NEC prevention.

While human milk’s clinical benefit is undisputed, how its specific components and downstream microbial metabolites protect the preterm gut from NEC is still not fully understood. This manuscript summarizes the development and function of key intestinal components—epithelial cells, mucosal immune cells, and gut microbiota—that maintain homeostasis in preterm infants. We then explore how bioactive components in human milk and their microbial metabolites, acting beyond nutrition, critically modulate this immature gut. Their balanced activity supports mucosal integrity, while dysregulation contributes to NEC pathogenesis.

Ontogeny of the preterm gut: epithelial and immune development

The IEC monolayer in preterm neonates

The IEC monolayer, which forms the villi and crypt structures of the small intestine, are polarized columnar monolayers that provide innate protective mechanisms against pathogens. They are composed of different cell types differentiated by LGR5-positive stem cells: enterocytes, Paneth cells, goblet cells, neuroendocrine cells, and M cells (Figure 1). The anatomical components of the gastrointestinal tract are morphologically mature by around 22 weeks of gestation. However, their magnitude and functional capacity remain considerably lower than those of term neonates.

Figure 1 Chronological development of cellular components in human gut. IgA, immunoglobulin A.

Enterocytes are the most prevalent cells in the intestinal epithelium and are responsible for both nutrient absorption and barrier function, the latter maintained by the formation of tight junctions between enterocytes. Although tight junctions can be observed as early as approximately 10 weeks of gestation, the intestinal barrier of preterm neonates remains more permeable than that of full-term neonates. This increased permeability makes them more susceptible to microbial invasion (11). Paneth cells and goblet cells are critical components to mucosal barrier and intestinal homeostasis. Paneth cells reside in the small intestinal crypts adjacent to the stem cells and secrete a variety of antimicrobial and inflammatory molecules into the lumen of the crypt. Many of these molecules are similar to those secreted by neutrophils and serve to shape the composition of the microbiota and limit exposure of the monolayer (including the stem cells) to pathogens. The number and activity of Paneth cells significantly increase after 29 weeks of gestational age (GA), as demonstrated by the increased expression of common Paneth cell products such as defensins, lactoferrin, and lysozyme (11,12). The number of goblet cells has remained stable from 17 weeks of gestation, although goblet cell density and mucus layer thickness increase with advancing GA (13,14). Goblet cells secrete mucins and intestinal trefoil factor 3, which together form a mucus layer covering the apical surface of enterocytes. This mucus layer serves as a physical barrier that traps pathogens, limiting their access to the intestinal epithelium. The expression of mucins and trefoil factor 3 increases across gestation, paralleling the maturation of intestinal innate immune defense (13-15). Enteroendocrine cells, which account for less than 1% of IECs, are responsible for the production of gut hormones in response to nutritional signals to regulate intestinal motility and secretion. The amount of enteroendocrine cells does not differ between preterm and term neonates (13). Intestinal M cells are closely related to gut-associated lymphoid tissue, and their phagocytosis contributes to the presentation of antigens to immune cells (16).

Due to the normal disruption of gut development in the uterus, premature neonates are susceptible to inflammation because some innate immune receptors, such as TLR4, are expressed at high levels in the intestinal epithelium. TLR4, a central bacterial signaling receptor that plays a crucial immunomodulatory role in the development of NEC, is expressed at high levels in the premature gut due to its critical role in the regulation of normal gut differentiation and development; however, the Gram-negative bacteria that colonize the premature gut in the postnatal period activate the deleterious consequences of excessive TLR4 signaling, such as recruiting pro-inflammatory Th17 cells and the initiation of the inflammatory cascade reaction, which in aggregate lead to the development of NEC (4,17).

Immature mucosal immune responses in preterm neonates

The mucosal immune cells are mainly found in the Peyer’s patches, mesenteric lymph nodes and the lamina propria, and they include macrophages, dendritic cells (DCs), innate lymphoid cells (ILCs), intraepithelial lymphocytes (IELs), adaptive immune cells (T lymphocytes and IgA-producing plasma cells), and stromal cells (Figure 1). The interactions between colonizing microbiota and the mucosal immune system are required to induce tolerance and homeostatic immune responses to promote the eventual maturation of the mucosal immune system.

Monocytes in the peripheral blood can be recruited to the inflamed intestine by the chemokine receptor CCR2 and then differentiate into lamina propria macrophages. At approximately 12 weeks of gestation, macrophages begin to appear in the gut mucosa and express various pattern recognition receptors, such as TLRs, NOD-like receptors and RIG-like receptors, to identify antigens (18). Phagocytosis and the expression of costimulatory molecules increase with increasing GA, and phagocytic function gradually improves; due to the abrupt interruption of gut development with preterm birth, both phagocytosis and the expression of costimulatory molecules are impaired (1). In addition, preterm macrophages in the small intestine show strong immunoreactivity for proinflammatory cytokines including tumor necrosis factor (TNF)-α and interleukin (IL)-8 (19). Myeloidderived mucosal DCs are specialized antigen-presenting cells that phagocytize luminal antigens by expressing some pattern recognition receptors, such as TLR-5, which recognizes flagellin protein component. CD103-expressing intestinal DCs regulate intestinal homeostasis by driving chemokine receptor CCR9 and promoting naive T cells differentiation, whereas preterm DCs are less able to secrete cytokines, impairing the adaptive immunity of preterm neonates (1,20).

ILCs participate in postnatal mucosal immunity by orchestrating antimicrobial immunity and modulating inflammation response. IL-17 and IL-22 are produced by ILC3 and ILC2, respectively, which can be found in the human fetal intestine during the second-trimester (21). It has been shown that ILC inflammatory subsets are prevalent in the human maternal-fetal interface and play an important role in the pathological process of preterm labor; however, their specific role in the fetal gut needs to be further explored (21,22). IEL is distributed between IECs, in which about 90% of IELs express T cell receptors (TCR) (23). Neonatal TCRγδ⁺ IELs show stronger immuno-protection and immunoregulatory activities than TCRαβ⁺ IELs, and the analysis of non-NEC surgical control samples show that the proportion of TCRαβ⁺ IELs reach its lowest point at about 28–32 weeks of GA (23,24).

T cells and B cells can be found in the human fetal intestine at 11–16 weeks gestation, and regulatory T cells (Treg) appear around 12 weeks gestation. Qazi et al. characterized the conventional T-cell compartment of very preterm neonates by analyzing their peripheral blood and found that significant decreases in the percentage of total lymphocytes, CD4 + and CD8 + lymphocytes during the first weeks of life may be associated with an increased risk of infection (25). Notably, T-cell phenotypes exhibit tissue-specific patterns during fetal development: while the spleen and lymph nodes favor Treg and T helper 2 (Th2)-biased differentiation, the fetal gut is characterized by a predominance of T helper 1 (Th1)-polarized CD4+ cells (26,27). Treg cells account for 30–40% of CD4+ T cells in fetal tissues, and maintain a higher density in the intestine than other immune organs (27). Treg cells in preterm neonates is less suppressive than in full-term neonates in inhibiting polymerization between DCs and conventional T cells, possibly due to low expression of CTLA-4 and FOXP3 (28). Tissue-resident memory T cells and effector memory T cells, which are found in the intestinal mucosa, start to form at the end of the first trimester: Schreurs et al. demonstrated that TNF-α producing CD4+ effector memory T cells predominated in the human fetal intestines via single-cell RNA sequencing analysis; CD69-expressing tissue-resident memory T cells produce cytokines that contribute to intestinal epithelial growth in mucosal development (26,27). B cells are primarily distributed in the lamina propria, and secretory immunoglobulin A (sIgA) is the major antibody produced by plasma cells in intestinal mucosal tissues and plays a crucial role in establishing immune repertoire of the developing intestine. Preterm neonates have lower immunoglobulin G (IgG) levels due to disrupted placental transmission, but human milk produced sufficient sIgA to promote barrier protection against intestinal pathogens (1).

Breastfeeding-driven microbiota: dominant taxa and functional metabolites

Microbial colonization patterns in preterm neonates

Gut microbiota, which provides energy and nutrients for intestinal development, is characterized by low diversity and increased pathogenic bacteria in preterm neonates, and the bacterial load tends to be very low during the initial colonization stage (29). Current research predominantly indicates a dominance of Bacillota (formerly Firmicutes) and Pseudomonadota (formerly Proteobacteria) in preterm neonates, alongside low levels of Bacteroidota (formerly Bacteroidetes) and a predominance of pathobionts during the neonatal intensive care unit hospitalization period (29-31).

After birth, microbial diversity in these infants gradually increases, with significant associations observed with feeding type, delivery mode, and antibiotic exposure (32). Although bacterial communities in preterm neonates demonstrate predictable successional dynamics, substantial controversy persists in current literature regarding their developmental trajectories—specifically the transition pathways between dominant taxa. Gut microbiota with gradually increasing Bifidobacterium abundance is a key biomarker for infant microbial health. Its establishment typically begins around 30 weeks postmenstrual age (PMA) or 1 week after birth, irrespective of gestational maturity at birth (30,33). However, in preterm neonates, the relative abundance of Bifidobacterium remains significantly reduced compared to term infants throughout the hospitalization period (34). Notably, preterm neonates exhibit an accelerated progression toward a Clostridial-dominated profile as early as 33–36 weeks PMA—distinct from term infants, in whom Clostridial abundance emerges only after 4 months of age and increases during weaning due to enhanced carbohydrate metabolism (35,36). Prior observations also revealed that preterm infant gut microbiota consistently stratify into four discrete community states, each defined by the exclusive dominance of Staphylococcus, Klebsiella, Escherichia, or Enterococcus (34,37). Rao et al. propose that interactions between bacteria, and between bacteria and fungi—particularly exploitative ones—may drive changes in community structure although fungal populations display higher-amplitude fluctuations with less defined temporal structure (37). The rate of this progression in preterm neonates correlates most strongly with GA (38), and by postnatal day 30, very low birth weight small-for-gestational-age preterm neonates exhibit significant divergence in gut bacterial community structure compared to appropriate-for-gestational-age counterparts (39). However, conflicting evidence exists regarding key developmental drivers: while intrinsic biological maturity (GA and birth weight) shapes colonization dynamics, Thänert et al. demonstrated that extrinsic pharmacological exposures (antibiotic and non-antibiotic medications) exert a stronger influence on microbiome composition than maternal factors or baseline infant characteristics (40). Toubon et al. propose that the relative impact of these factors varies temporally and depends on sampling windows, offering a possible explanation for these apparent contradictions (30).

Human milk oligosaccharides (HMOs) are small sugar molecules produced in abundance by the mother. They are not digestible by the infant; instead, their primary purpose is to shape the infant gut microbiota, as they serve as a source of nutrition for only limited bacterial taxa, predominantly Bifidobacterium and Bacteroides species. Masi et al. demonstrated that alterations in the concentrations of specific HMO components are linked to changes in microbiota development (41). Human milk facilitates maternal microbial transfer, promoting colonization by beneficial bacteria and enhancing microbiome diversity. Significant divergences emerge in microbial community structure, bacterial diversity, and genus-level abundances between human milk- and formula-fed infants. The abundance of Enterobacteriaceae is approximately 3-fold higher in infants fed any formula compared to breast-fed infants (42), and an association is also observed between reduced human milk feeding and microbiota types predominantly composed of Staphylococcus epidermidis or Enterobacteriaceae (43). In addition, human milk promotes enrichment of beneficial Lactobacillales and Bacillales, with other studies confirming superior colonization by Bifidobacterium and Lactobacillus within 2–3 weeks after birth (44).

Microbiota-derived metabolites in preterm neonates

Current clinical evidence predominantly supports that the gut microbiota of breastfed preterm neonates is enriched with specific commensals (e.g., Bifidobacterium, Lactobacillus, Clostridiales species) exhibiting enhanced capacity for fecal short-chain fatty acids (SCFAs) production (45,46). Bifidobacterium, which is predominant in the gut of breast-fed infants, are the main microbial sources of SCFAs (42,47), due to its rich glycosyl hydrolase genome that efficiently utilizes HMOs. Consistent with the ability of supplemental Bifidobacterium strains to metabolize HMOs to acetate, Fecal acetate and propionate concentrations are higher in breast-fed infants than in formula-fed infants and formula-fed infants had an impaired ability to metabolize HMOs (48). Notably, the ability of Bifidobacterium spp. to utilize HMOs is strain-specific (41).

The impact of breastfeeding versus formula feeding on SCFA profiles in preterm neonates remains controversial. Chen et al. observed elevated intestinal SCFA concentrations (predominantly acetate, butyrate, and propionate) in formula-fed preterm neonates, based on fecal samples from 60 neonates during the first postnatal month (49). Conversely, others reported higher levels of total SCFAs, acetate, and propionate in breastfed infants (50). This discrepancy may arise from methodological variations—notably, unaccounted confounding factors such as antibiotic exposure and delivery mode, which significantly modulate gut microbial composition and metabolic activity.

Laursen et al. discovered that specific Bifidobacterium strains promoted by breastfeeding—particularly B. longum, B. bifidum, and B. breve – metabolize tryptophan into indolelactic acid using a newly identified enzyme, aromatic lactate dehydrogenase (51). The link between HMOs and tryptophan metabolism is explained by the fact that specific bacterial strains co-express high levels of genes involved in both HMO degradation and aromatic lactate dehydrogenase. When HMOs are added to the bacterial culture medium, they increase the production of indolelactic acid. This metabolite exerts dual immunomodulatory effects: on one hand, it activates the aryl hydrocarbon receptor (AHR) on T cells to promote IL-22 expression, thereby enhancing gut barrier integrity and pathogen defense; on the other hand, it suppresses pro-inflammatory IL-12p70 in macrophages by activating both AHR and hydroxycarboxylic acid receptor 3, helping to mitigate excessive inflammation (51).

Mechanisms of human milk bioactive components in NEC

Human milk-derived macronutrients—proteins, lipids, and carbohydrates—serve as foundational substrates for intestinal development while actively mitigating NEC through distinct mechanistic pathways (Table 1, Figure 2).

Figure 2 Mechanisms of human milk bioactive components and microbiota-derived metabolites in necrotizing enterocolitis. This schematic illustrates the synergistic defense against NEC in preterm infants through human milk-derived components and their microbial metabolites. Within the gut lumen, milk proteins enhance macrophage phagocytosis, stimulate IEC proliferation via Wnt/Ras-MAPK pathways, and stimulate DC cell differentiation. Lipids, including MFGM components, block bacterial adhesion, and repair barrier integrity, while structured fatty acids balance TLR4/NFκB signaling and T-cell polarization. HMOs act as decoy receptors to prevent pathogen binding, suppress CD14/TLR4 activation, and promote Treg differentiation. These bioactives collectively shape a beneficial microbiota dominated by Bifidobacterium/Lactobacillus, which metabolize milk components to generate protective SCFAs and tryptophan derivatives. SCFAs strengthen the epithelial barrier through occludin/MUC2 upregulation, inhibit ERK1/2 pathways to expand Tregs. Tryptophan metabolites activate AHR, suppressing STAT1/TLR4 hyperresponsiveness and cytokine storms. AHR, aryl hydrocarbon receptor; DC, dendritic cell; eNOS, endothelial nitric oxide synthase; HMOs, human milk oligosaccharides; IEC, intestinal epithelial cell; IL, interleukin; ILA, indole-3-lactic acid; LCPUFA, long-chain polyunsaturated fatty acid; MFGM, milk fat globule membrane; NEC, necrotizing enterocolitis; NF-κB, nuclear factor kappa-B; SCFAs, short-chain fatty acids; STAT1, signal transducer and activator of transcription 1; TLR4, Toll-like receptor 4; Treg, regulatory T cell.

Proteins

Human milk proteins, primarily comprising casein and whey protein, play a vital role in protecting preterm neonates against NEC.

Casein exists in several genetic variants. Its derivative, κ-casein-derived glycomacropeptide (GMP), exhibits immunomodulatory and anti-inflammatory properties in experimental models: GMP enhances macrophage phagocytic activity and reduces gut IL-17 and TNF-α levels by modulating Th17 cells (52). However, in preterm piglets, GMP supplementation improves intestinal lactase activity and reduces LPS-induced IL-8 secretion in IECs, but shows no significant effects on villus structure, microbiota, or systemic immunity, suggesting its mucosal immunomodulatory effects may be limited (53). Whey protein, rich in bioactive components like lactoferrin, α-lactalbumin, β-lactoglobulin, immunoglobulins, and osteopontin, demonstrates significant protective potential. Lactoferrin, the most abundant whey protein, is increasingly recognized for its multifaceted role in NEC prevention: it plays a direct anti-microbial role via specifically binding to iron and inhibiting the proliferation of intestinal microbiota that depend on iron for growth (54); it upregulates the differentiation of monocytes to tolerogenic DCs in a concentration-dependent manner through different types of lectin (i.e., intestinal lactoferrin receptor) that are widely distributed in intestinal immune cells, and inhibits TLR-4-mediated inflammatory response (55); lactoferrin not only induces the proliferation and differentiation of mucosal T lymphocytes (i.e., increased the number of NK, CD4+ and CD8+ cells), but also stimulates the production of protective cytokines and increases the concentration of plasma IgG, IgA and IgM in neonatal piglets (56,57); lactoferrin stimulates the differentiation of intestinal epithelial stem cells and the proliferation of IECs and intestinal Peyer’s patches, and enhances intestinal crypt proliferation and depth to maintain intestinal barrier integrity via Wnt signaling and Ras-MAPK pathway (58); Crucially, a review of clinical trials found that enteral lactoferrin supplementation significantly reduced late onset sepsis in preterm neonates, but did not demonstrate a decrease in NEC; however, when combined with probiotics, one study suggested a potential reduction in NEC, but the evidence is of very low certainty (92).

Furthermore, key amino acids found in human milk, such as glutamine and arginine (and their precursors), contribute synergistically to NEC protection through anti-inflammatory and antioxidant mechanisms: glutamine stimulates the differentiation of intestinal stem cells primarily towards goblet cells, as evidenced by increased Muc2 expression in the crypts, and it may induce the secretion of sIgA via intestinal microorganisms and T cell-dependent or T cell-independent pathways, and promoted the anti-apoptotic activity of enterocytes through down-regulation of cytochrome-dependent casepase-3 expression and glutathione-related antioxidant effect (59,60); Nitric oxide produced by arginine metabolism protects the gut via preventing ischemic injury, stimulates intestinal protein synthesis and increases IECs proliferation and growth through activated mammalian target of rapamycin and p70S6 kinase signaling (61,62). Whether and how this occurs in humans is unclear. The potential impact of glutamine‑containing peptides may also be important, and recent studies suggest that glutamine peptides protect the intestinal barrier by regulating tight junctions, mucin production, inflammatory responses, and gut microbiota (93).

Lipids and fatty acids

Triglycerides (TGs), the core component of human milk fat globules (MFGs), deliver half of the non-protein energy in enteral nutrition. However, the preterm gut’s immaturity—marked by deficient lipase activity (notably bile salt-stimulated lipase) and poor IEC absorptive capacity—leads to TG accumulation in the distal intestine (63,64). This malabsorption fuels NEC pathogenesis by triggering reactive oxygen species generation, oxidative stress, and inflammation, with recent human evidence implicating ferroptosis driven by lipid peroxidation (64,65). The bioactive milk fat globule membrane (MFGM) enveloping TGs originates from maternal epithelial cells within the alveoli of the mammary gland and it acts as a multifaceted shield: MFGM components stimulate Paneth cell and goblet cell proliferation while repairing tight junctions (66); MFGM surface molecules like mucin 1 and DC-specific intercellular adhesion molecule-3 block pathogen adherence to DCs; butyrophilin activates intestinal TCRαβ⁺/γδ⁺ lymphocytes; and MFG-EGF factor 8 enhances macrophage phagocytosis to dampen inflammation (64,67).

While pro-inflammatory saturated fatty acids (SFAs) like lauric and stearic acid induce mucosal damage via TLR4/NF-κB/COX-2 signaling in macrophages (68), the structured SFA β-palmitic acid promotes intestinal homeostasis by upregulating Foxp3, Ebi3, and antioxidant genes to bolster Treg-mediated anti-inflammatory responses (69). Specific fatty acid deficiencies can also lead to an increased risk of NEC. During fetal life, long-chain polyunsaturated fatty acids (LCPUFAs) are acquired mainly through placental transfer, which peaks during the third trimester, and systemic LCPUFA levels and the storage of adipose tissue are reduced due to preterm birth interruptions (64). Docosahexaenoic acid (DHA) and arachidonic acid (AA) are important components of LCPUFAs and play a critical role in regulating inflammation during early development: DHA and AA supplementation decreases the IL-1R1 receptor and NF-κB1 gene expression in IECs, blocks the platelet activating factor-induced TLR4 expression, and enhances the Th cells responses (64,70). The imbalance in n-3 to n-6 LCPUFAs may affect intestinal permeability and inflammatory pathways and inhibit Lactobacillus and Bifidobacterium, which are essential for mucus production, tight junction formation and Th1/Th2 balance in a dose-dependent manner (71,72). In addition, Singh et al. demonstrated reduced expression of innate immune response genes, such as TLR9, MUC2 and tight junction proteins, in fat-1 transgenic mice with significantly higher n-6 LCPUFAs than n-3 LCPUFAs. AA: DHA ratio >1.0 is also demonstrated to increase the height of ileal villi and muscular thickness (73). Despite compelling preclinical data, clinical trials of LCPUFA supplementation show mixed and limited effects on NEC risk (71,94). This discrepancy stems from heterogeneous study designs: trials lacked NEC-focused protocols, varied in LCPUFA doses/ratios, and failed to control baseline diets or feeding strategies. Rigorous clinical investigations addressing these limitations are urgently needed to translate mechanistic insights into effective nutritional interventions.

Carbohydrates

The role of carbohydrates in the development of NEC has not been deeply explored, and the current research focuses on the effects of lactose and HMOs, the two most abundant carbohydrates in human milk, on NEC.

Lactose, reaching concentrations up to 70 g/L, demonstrates superior absorbability in preterm pigs compared to maltodextrin-based formulas, correlating with reduced NEC incidence (95). This effect is attributed in part to the significantly higher hydrolytic activity of lactase compared to maltases in the piglet small intestine. However, caution is warranted when extrapolating these findings to preterm neonates, as piglets have substantially lower maltase activity than human infants and thus may be uniquely susceptible to maldigestion and NEC when fed maltodextrin-based formulas (96). Far beyond nutrition, HMOs—the third most abundant component in human milk (≈17 g/L in colostrum, 11 g/L in mature milk)—serve as potent immunomodulators. Structurally mimicking host cell glycans, HMOs act as decoy receptors, blocking pathogenic adhesion to IECs and suppressing proinflammatory cytokine expression (74). Their immunoregulatory functions extend through multiple pathways: HMOs enhance Th1-polarized responses and promote Treg cell conversion during early mucosal immune development, partly by amplifying TLR9 agonist effects (75,76); they attenuate LPS-induced IL-8 release via CD14 inhibition and reduce endothelial nitric oxide synthase expression to restore mesenteric perfusion (77,78); and they upregulate genes critical for intestinal transport, absorption, and secretion while promoting monocyte differentiation and limiting neutrophil infiltration via selectin inhibition (79). Clinically, formula supplementation with specific HMOs (e.g., 2′-fucosyllactose, disialyllacto-N-tetraose) confirms their immunoprotective effects, and intriguingly, HMO profiles in human milk may serve as non-invasive biomarkers for NEC risk stratification (80).

Mechanisms of human milk’s microbial metabolites in NEC

It may be the function of gut microbes (what they are doing), rather than just the composition (which gut microbes are present or predominant), that is important in NEC pathogenesis. Microbial function includes secretion of metabolites, expression of other gene products, and even such simple effects as alteration of the pH and oxygen content of the intestinal lumen. A systematic review by Pammi et al. revealed that preterm neonates developing NEC exhibit gut dysbiosis (characterized by increased Proteobacteria and decreased Firmicutes/Bacteroidetes)—a profile directly opposing the Firmicutes- and Bacteroidetes-dominant microbiome typically seen in breastfed preterm neonates (97). Warner et al. further refined this pattern in infants <27 weeks gestation, reporting a significant temporal rise in Gammaproteobacteria (a Proteobacteria class) with concurrent declines in Negativicutes and combined Clostridia-Negativicutes classes (98). However, integrated analyses of fecal sequencing and metabolomics challenge the existence of a universal microbial signature for NEC. These studies propose that instability in gut microbiome development—rather than specific taxonomic shifts—may act as the primary trigger, initiating and perpetuating the exaggerated inflammatory response characteristic of NEC (99).

SCFAs and NEC

Preterm neonates with NEC exhibit significantly reduced levels of major SCFAs like butyrate, propionate, and acetate (40). These major SCFAs demonstrate potent anti-inflammatory and protective effects in the immature gut, likely through suppression of the ERK1/2 pathway (81), thereby ameliorating inflammation in the fetal small intestine (82). The well-established protective role of butyrate in NEC directly aligns with clinical observations: this key microbial metabolite—predominantly produced by Firmicutes and Bacteroidetes—is depleted in NEC infants, mirroring the reduced abundance of these bacterial phyla preceding disease onset (83,97). In vivo, butyrate enhances Treg populations to promote immune tolerance, and reduces macrophage inflammatory protein2 secretion (83,84); butyrate induces the expression of single immunoglobulin interleukin-1-related receptor and A20, which repress experimental NEC (85). Concurrently, in vitro studies using human immature enterocytes demonstrate that butyrate strengthens intestinal barrier integrity by upregulating tight junction proteins and mucin gene expression (83). Furthermore, butyrate transduces anti-inflammatory signals via G-protein coupled receptors and epigenetic regulation (HDAC3/HDAC5 inhibition) (86).

Tryptophan metabolites and NEC

Human milk is rich in some bacteria, including Bifidobacterium, Bacteroides, and Clostridium, which have the ability to convert tryptophan into indole and its derivatives (100). Evidence indicates that the gut microbiota of preterm neonates diagnosed with NEC exhibits significant depletion of bacterial enzymes critical for tryptophan metabolism (101), and the L-tryptophan degradation pathway in NEC infants is also upregulated at the time of NEC onset (102).

As a primary receptor for tryptophan metabolites, AHR plays a crucial protective role against NEC. Activation of the AHR by indole-3-carbinole in the intestinal epithelium or CD11c+ cells protects neonatal mice from NEC via reducing TLR4 signaling (87,88). Indole-3-lactic acid (ILA)—another major tryptophan metabolite in human milk produced by Bifidobacterium longum subsp. infantis—suppresses inflammatory responses by downregulating the signal transducer and activator of transcription 1 (STAT1) signaling pathway (81). Consistent with this, a clinical study comparing preterm infants given B. infantis versus those given L. reuteri (which does not consume HMOs or produce ILA) found that the B. infantis group had lower fecal HMOs (implying consumption by gut microbes), greater fecal ILA (implying production by gut microbes), and lower fecal calprotectin (a marker of gut inflammation) (89). In addition, Lu et al. also demonstrated that human milk is rich in maternally derived AHR ligands (87). Collectively, these findings illuminate a dual-defense mechanism wherein human milk not only directly supplies protective AHR ligands but also fosters Bifidobacterium-dependent generation of tryptophan metabolites, cooperatively constraining TLR4-driven inflammation and STAT1-mediated hyperresponsiveness—a synergistic protection eroded in NEC by microbial enzymatic depletion.

Stewart et al. identified that four of five metabolites most predictive of NEC were linked to microbiome-mediated linoleate metabolism (99), and the gut microbiota (including Lactobacillus, Clostridium butyricum, Bifidobacterium, and the family Ruminococcaceae)-regulated linoleic acid pathway, a target metabolic product that determines the degree of inflammation, regulates Th17 differentiation through the AHR while promoting Treg cell differentiation through phosphorylation of Stat1 (90,91).

Conclusions

NEC is a heterogeneous, multifactorial disease which is characterized by epithelial barrier dysfunction, impaired mucosal immunity, and dysbiotic microbial colonization. Human milk exerts protective effects against NEC through synergistic mechanisms: primarily, its bioactive components enhance barrier integrity, suppress pro-inflammatory pathways, and modulate immune cell function; secondarily, human milk shapes a protective gut microbiota enriched with Bifidobacterium and Lactobacillus. These commensals generate key metabolites that reinforce mucosal homeostasis by promoting Treg differentiation, enhancing tight junction expression, and antagonizing pro-inflammatory pathways. Current evidence underscores the critical role of human milk bioactives and the microbiome-metabolite axis in preventing NEC. Future NEC prevention should leverage these insights through targeted nutrient supplementation and next-generation probiotics designed to enhance production of SCFAs and indole derivatives.

Acknowledgments

None.

Footnote

Peer Review File: Available at https://tp.amegroups.com/article/view/10.21037/tp-2026-0311/prf

Funding: This study was supported by National Natural Science Foundation of China (No. #82371712, to M.H.).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tp.amegroups.com/article/view/10.21037/tp-2026-0311/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. Sampah MES, Hackam DJ. Dysregulated Mucosal Immunity and Associated Pathogeneses in Preterm Neonates. Front Immunol 2020;11:899. [Crossref] [PubMed]
2. Ren S, Pan X, Hui Y, et al. Subclinical necrotizing enterocolitis-induced systemic immune suppression in neonatal preterm pigs. Am J Physiol Gastrointest Liver Physiol 2021;321:G18-28. [Crossref] [PubMed]
3. Zhang D, Xie D, Qu Y, et al. Digging deeper into necrotizing enterocolitis: bridging clinical, microbial, and molecular perspectives. Gut Microbes 2025;17:2451071. [Crossref] [PubMed]
4. Hackam DJ, Sodhi CP. Bench to bedside - new insights into the pathogenesis of necrotizing enterocolitis. Nat Rev Gastroenterol Hepatol 2022;19:468-79. [Crossref] [PubMed]
5. Cuna A, Morowitz MJ, Sampath V. Early antibiotics and risk for necrotizing enterocolitis in premature infants: A narrative review. Front Pediatr 2023;11:1112812. [Crossref] [PubMed]
6. Roberts AG, Younge N, Greenberg RG. Neonatal Necrotizing Enterocolitis: An Update on Pathophysiology, Treatment, and Prevention. Paediatr Drugs 2024;26:259-75. [Crossref] [PubMed]
7. Ma F, Hao H, Gao X, et al. Melatonin ameliorates necrotizing enterocolitis by preventing Th17/Treg imbalance through activation of the AMPK/SIRT1 pathway. Theranostics 2020;10:7730-46. [Crossref] [PubMed]
8. Altobelli E, Angeletti PM, Verrotti A, et al. The Impact of Human Milk on Necrotizing Enterocolitis: A Systematic Review and Meta-Analysis. Nutrients 2020;12:1322. [Crossref] [PubMed]
9. Filatava EJ, Liu Z, Xie J, et al. The preterm human milk microbiota fluctuates by postpartum week and is characterized by gestational age and maternal BMI. mBio 2023;14:e0210623. [Crossref] [PubMed]
10. Masi AC, Beck LC, Perry JD, et al. Human milk microbiota, oligosaccharide profiles, and infant gut microbiome in preterm infants diagnosed with necrotizing enterocolitis. Cell Rep Med 2024;5:101708. [Crossref] [PubMed]
11. Frazer LC, Good M. Intestinal epithelium in early life. Mucosal Immunol 2022;15:1181-7. [Crossref] [PubMed]
12. Sankova MV, Nikolenko VN, Bolotskaia AA, et al. Paneth Cells: Recent Updates on Elucidating Therapeutic Implications in Gastroenterological Disease Management. Curr Med Chem 2025;32:9679-706. [Crossref] [PubMed]
13. Stanford AH, Gong H, Noonan M, et al. A direct comparison of mouse and human intestinal development using epithelial gene expression patterns. Pediatr Res 2020;88:66-76. [Crossref] [PubMed]
14. Demers-Mathieu V. The immature intestinal epithelial cells in preterm infants play a role in the necrotizing enterocolitis pathogenesis: A review. Health Sciences Review 2022;4:100033.
15. Yu Y, Lu L, Sun J, et al. Preterm infant gut microbiota affects intestinal epithelial development in a humanized microbiome gnotobiotic mouse model. Am J Physiol Gastrointest Liver Physiol 2016;311:G521-32. [Crossref] [PubMed]
16. Pracht K, Wittner J, Kagerer F, et al. The intestine: A highly dynamic microenvironment for IgA plasma cells. Front Immunol 2023;14:1114348. [Crossref] [PubMed]
17. Huang D, Wang P, Chen J, et al. Selective targeting of MD2 attenuates intestinal inflammation and prevents neonatal necrotizing enterocolitis by suppressing TLR4 signaling. Front Immunol 2022;13:995791. [Crossref] [PubMed]
18. Michalski C, Cheung C, Oh JH, et al. DDIT4L regulates mitochondrial and innate immune activities in early life. JCI Insight 2024;9:e172312. [Crossref] [PubMed]
19. Shen L, Zhong X, Ji H, et al. Macrophage α7nAChR alleviates the inflammation of neonatal necrotizing enterocolitis through mTOR/NLRP3/IL-1β pathway. Int Immunopharmacol 2024;139:112590. [Crossref] [PubMed]
20. Yoon G, Davidson LA, Goldsby JS, et al. Exfoliated epithelial cell transcriptome reflects both small and large intestinal cell signatures in piglets. Am J Physiol Gastrointest Liver Physiol 2021;321:G41-51. [Crossref] [PubMed]
21. Tang LC, Xu XH, Jin LP. Molecular characteristics and possible functions of innate lymphoid cells in the uterus and gut. Cytokine Growth Factor Rev 2020;52:15-24. [Crossref] [PubMed]
22. Xu Y, Romero R, Miller D, et al. Innate lymphoid cells at the human maternal-fetal interface in spontaneous preterm labor. Am J Reprod Immunol 2018;79:e12820. [Crossref] [PubMed]
23. Wiemers TC, Riedel J, Dressler N, et al. Murine intestinal epithelial cells and lymphocytes undergo contrasting inflammatory shifts during gastrointestinal development. Pediatr Res 2026;99:1128-36. [Crossref] [PubMed]
24. Venkatraman A, Perry JM, Sampath V. Temporal mapping of epithelial vs. immune cell inflammatory shifts in the neonatal intestinal mucosa. Pediatr Res 2026;99:839-40.
25. Qazi KR, Bach Jensen G, van der Heiden M, et al. Extremely Preterm Infants Have Significant Alterations in Their Conventional T Cell Compartment during the First Weeks of Life. J Immunol 2020;204:68-77. [Crossref] [PubMed]
26. Schreurs RRCE, Baumdick ME, Sagebiel AF, et al. Human Fetal TNF-α-Cytokine-Producing CD4(+) Effector Memory T Cells Promote Intestinal Development and Mediate Inflammation Early in Life. Immunity 2019;50:462-476.
27. Thome JJ, Bickham KL, Ohmura Y, et al. Early-life compartmentalization of human T cell differentiation and regulatory function in mucosal and lymphoid tissues. Nat Med 2016;22:72-7. [Crossref] [PubMed]
28. Verma S, Bradley MC, Gray J, et al. Distinct Localization, Transcriptional Profiles, and Functionality in Early Life Tonsil Regulatory T Cells. J Immunol 2024;213:306-16. [Crossref] [PubMed]
29. Healy DB, Ryan CA, Ross RP, et al. Clinical implications of preterm infant gut microbiome development. Nat Microbiol 2022;7:22-33. [Crossref] [PubMed]
30. Toubon G, Patin C, Delannoy J, et al. Very preterm gut microbiota development from the first week of life to 3.5 years of age: a prospective longitudinal multicenter study. Microbiol Spectr 2025;13:e0163624. [Crossref] [PubMed]
31. Tauchi H, Yahagi K, Yamauchi T, et al. Gut microbiota development of preterm infants hospitalised in intensive care units. Benef Microbes 2019;10:641-51. [Crossref] [PubMed]
32. Jia Q, Yu X, Chang Y, et al. Dynamic Changes of the Gut Microbiota in Preterm Infants With Different Gestational Age. Front Microbiol 2022;13:923273. [Crossref] [PubMed]
33. Korpela K, Blakstad EW, Moltu SJ, et al. Intestinal microbiota development and gestational age in preterm neonates. Sci Rep 2018;8:2453. [Crossref] [PubMed]
34. Toubon G, Butel MJ, Rozé JC, et al. Very Preterm Children Gut Microbiota Comparison at the Neonatal Period of 1 Month and 3.5 Years of Life. Front Microbiol 2022;13:919317. [Crossref] [PubMed]
35. Zeng S, Wang H, Zhang L, et al. The Gut Resistome Atlas in Preterm Infants Enables Prediction of Necrotizing Enterocolitis Onset. Adv Sci (Weinh) 2025;12:e05154. [Crossref] [PubMed]
36. Beller L, Deboutte W, Falony G, et al. Successional Stages in Infant Gut Microbiota Maturation. mBio 2021;12:e0185721. [Crossref] [PubMed]
37. Rao C, Coyte KZ, Bainter W, et al. Multi-kingdom ecological drivers of microbiota assembly in preterm infants. Nature 2021;591:633-8. [Crossref] [PubMed]
38. Moriel N, Jones L, Harpenas E, et al. Development of the preterm infant gut and gastric residuals microbiome. Nat Commun 2025;16:9848. [Crossref] [PubMed]
39. Chang HY, Chiang Chiau JS, Chang JH, et al. Characteristics of Gut Microbiota in Small for Gestational Age Infants with Very Low Birth Weight. Nutrients 2022;14:5158. [Crossref] [PubMed]
40. Thänert R, Schwartz DJ, Keen EC, et al. Clinical sequelae of gut microbiome development and disruption in hospitalized preterm infants. Cell Host Microbe 2024;32:1822-1837.
41. Masi AC, Embleton ND, Lamb CA, et al. Human milk oligosaccharide DSLNT and gut microbiome in preterm infants predicts necrotising enterocolitis. Gut 2021;70:2273-82. [Crossref] [PubMed]
42. Pärnänen KMM, Hultman J, Markkanen M, et al. Early-life formula feeding is associated with infant gut microbiota alterations and an increased antibiotic resistance load. Am J Clin Nutr 2022;115:407-21. [Crossref] [PubMed]
43. Lemme-Dumit JM, Song Y, Lwin HW, et al. Altered Gut Microbiome and Fecal Immune Phenotype in Early Preterm Infants With Leaky Gut. Front Immunol 2022;13:815046. [Crossref] [PubMed]
44. Natal ACC, de Paula Menezes R, de Brito Röder DVD. Role of maternal milk in providing a healthy intestinal microbiome for the preterm neonate. Pediatr Res 2025;97:2235-42. [Crossref] [PubMed]
45. Markowiak-Kopeć P, Śliżewska K. The Effect of Probiotics on the Production of Short-Chain Fatty Acids by Human Intestinal Microbiome. Nutrients 2020;12:1107. [Crossref] [PubMed]
46. Cong X, Xu W, Janton S, et al. Gut Microbiome Developmental Patterns in Early Life of Preterm Infants: Impacts of Feeding and Gender. PLoS One 2016;11:e0152751. [Crossref] [PubMed]
47. Beck LC, Masi AC, Young GR, et al. Strain-specific impacts of probiotics are a significant driver of gut microbiome development in very preterm infants. Nat Microbiol 2022;7:1525-35. [Crossref] [PubMed]
48. Neumann CJ, Mahnert A, Kumpitsch C, et al. Clinical NEC prevention practices drive different microbiome profiles and functional responses in the preterm intestine. Nat Commun 2023;14:1349. [Crossref] [PubMed]
49. Chen C, Yin Q, Wu H, et al. Different Effects of Premature Infant Formula and Breast Milk on Intestinal Microecological Development in Premature Infants. Front Microbiol 2019;10:3020. [Crossref] [PubMed]
50. Kukaev E, Krogh-Jensen O, Starodubtseva N, et al. Longitudinal Fecal Short-Chain Fatty Acid Trajectories in Preterm Infants with Early-Onset Neonatal Sepsis: A Pilot Study. Life (Basel) 2025;15:1943. [Crossref] [PubMed]
51. Laursen MF, Sakanaka M, von Burg N, et al. Bifidobacterium species associated with breastfeeding produce aromatic lactic acids in the infant gut. Nat Microbiol 2021;6:1367-82. [Crossref] [PubMed]
52. Qu Y, Park SH, Dallas DC. Evaluating the Potential of Casein Glycomacropeptide in Adult Irritable Bowel Syndrome Management: A Pilot Study. Nutrients 2023;15:4174. [Crossref] [PubMed]
53. Ren S, Hui Y, Goericke-Pesch S, et al. Gut and immune effects of bioactive milk factors in preterm pigs exposed to prenatal inflammation. Am J Physiol Gastrointest Liver Physiol 2019;317:G67-77. [Crossref] [PubMed]
54. Peña-Juárez MC, Guadarrama-Escobar OR, Serrano-Castañeda P, et al. Synergistic Effect of Retinoic Acid and Lactoferrin in the Maintenance of Gut Homeostasis. Biomolecules 2024;14:78. [Crossref] [PubMed]
55. Perdijk O, van Neerven RJJ, van den Brink E, et al. Bovine Lactoferrin Modulates Dendritic Cell Differentiation and Function. Nutrients 2018;10:848. [Crossref] [PubMed]
56. Wu H, Fan L, Gao Y, et al. The Protective Effects of Iron Free Lactoferrin on Lipopolysaccharide-Induced Intestinal Inflammatory Injury via Modulating the NF-κB/PPAR Signaling Pathway. Foods 2022;11:3378. [Crossref] [PubMed]
57. Lyra S, Groß-Weege I, Leonhardt S, et al. editors. Real-Time Respiration Monitoring of Neonates from Thermography Images Using Deep Learning. Image Analysis and Processing – ICIAP 2022. Cham: Springer International Publishing, 2022.
58. Jiang R, Du X, Lönnerdal B. Effects of Forming Lactoferrin-Milk Protein Complexes on Lactoferrin Functionality and Intestinal Development in Infancy. Nutrients 2024;16:4077. [Crossref] [PubMed]
59. Wu M, Xiao H, Liu G, et al. Glutamine promotes intestinal SIgA secretion through intestinal microbiota and IL-13. Mol Nutr Food Res 2016;60:1637-48. [Crossref] [PubMed]
60. Chen Y, Tseng SH, Yao CL, et al. Distinct Effects of Growth Hormone and Glutamine on Activation of Intestinal Stem Cells. JPEN J Parenter Enteral Nutr 2018;42:642-51. [Crossref] [PubMed]
61. Robinson JL, Smith VA, Stoll B, et al. Prematurity reduces citrulline-arginine-nitric oxide production and precedes the onset of necrotizing enterocolitis in piglets. Am J Physiol Gastrointest Liver Physiol 2018;315:G638-49. [Crossref] [PubMed]
62. Wang X, Zhang T, Li W, et al. Arginine alleviates Clostridium perfringens α toxin-induced intestinal injury in vivo and in vitro via the SLC38A9/mTORC1 pathway. Front Immunol 2024;15:1357072. [Crossref] [PubMed]
63. Indrio F, Neu J, Pettoello-Mantovani M, et al. Development of the Gastrointestinal Tract in Newborns as a Challenge for an Appropriate Nutrition: A Narrative Review. Nutrients 2022;14:1405. [Crossref] [PubMed]
64. Alshaikh BN, Reyes Loredo A, Knauff M, et al. The Role of Dietary Fats in the Development and Prevention of Necrotizing Enterocolitis. Nutrients 2021;14:145. [Crossref] [PubMed]
65. Dang D, Zhang C, Meng Z, et al. Integrative analysis links ferroptosis to necrotizing enterocolitis and reveals the role of ACSL4 in immune disorders. iScience 2022;25:105406. [Crossref] [PubMed]
66. Bhinder G, Allaire JM, Garcia C, et al. Milk Fat Globule Membrane Supplementation in Formula Modulates the Neonatal Gut Microbiome and Normalizes Intestinal Development. Sci Rep 2017;7:45274. [Crossref] [PubMed]
67. Ren Y, Lu Y, Cui Q, et al. Milk fat globule-EGF factor 8/ATP-binding cassette subfamily E member 1 axis maintains mitophagy flux homeostasis to suppress ferroptosis in acute pancreatitis. Clin Transl Med 2026;16:e70619. [Crossref] [PubMed]
68. Sun L. Low-dose cyclooxygenase-2 (COX-2) inhibitor celecoxib plays a protective role in the rat model of neonatal necrotizing enterocolitis. Bioengineered 2021;12:7234-45. [Crossref] [PubMed]
69. Burge K, Vieira F, Eckert J, et al. Lipid Composition, Digestion, and Absorption Differences among Neonatal Feeding Strategies: Potential Implications for Intestinal Inflammation in Preterm Infants. Nutrients 2021;13:550. [Crossref] [PubMed]
70. Wijendran V, Brenna JT, Wang DH, et al. Long-chain polyunsaturated fatty acids attenuate the IL-1β-induced proinflammatory response in human fetal intestinal epithelial cells. Pediatr Res 2015;78:626-33. [Crossref] [PubMed]
71. Ramiro-Cortijo D, Singh P, Liu Y, et al. Breast Milk Lipids and Fatty Acids in Regulating Neonatal Intestinal Development and Protecting against Intestinal Injury. Nutrients 2020;12:534. [Crossref] [PubMed]
72. Akinsulire O, Perides G, Anez-Bustillos L, et al. Early Enteral Administration of a Complex Lipid Emulsion Supplement Prevents Postnatal Deficits in Docosahexaenoic and Arachidonic Acids and Increases Tissue Accretion of Lipophilic Nutrients in Preterm Piglets. JPEN J Parenter Enteral Nutr 2020;44:69-79. [Crossref] [PubMed]
73. Singh P, Ochoa-Allemant P, Brown J, et al. Effect of polyunsaturated fatty acids on postnatal ileum development using the fat-1 transgenic mouse model. Pediatr Res 2019;85:556-65. [Crossref] [PubMed]
74. Wu RY, Li B, Horne RG, et al. Structure-Function Relationships of Human Milk Oligosaccharides on the Intestinal Epithelial Transcriptome in Caco-2 Cells and a Murine Model of Necrotizing Enterocolitis. Mol Nutr Food Res 2022;66:e2100893. [Crossref] [PubMed]
75. Zuurveld M, Ayechu-Muruzabal V, Folkerts G, et al. Specific Human Milk Oligosaccharides Differentially Promote Th1 and Regulatory Responses in a CpG-Activated Epithelial/Immune Cell Coculture. Biomolecules 2023;13:263. [Crossref] [PubMed]
76. Ayechu-Muruzabal V, van de Kaa M, Mukherjee R, et al. Modulation of the Epithelial-Immune Cell Crosstalk and Related Galectin Secretion by DP3-5 Galacto-Oligosaccharides and β-3'Galactosyllactose. Biomolecules 2022;12:384. [Crossref] [PubMed]
77. Good M, Sodhi CP, Yamaguchi Y, et al. The human milk oligosaccharide 2'-fucosyllactose attenuates the severity of experimental necrotising enterocolitis by enhancing mesenteric perfusion in the neonatal intestine. Br J Nutr 2016;116:1175-87. [Crossref] [PubMed]
78. Verkhnyatskaya SA, Kong C, Klostermann CE, et al. Digestion, fermentation, and pathogen anti-adhesive properties of the hMO-mimic di-fucosyl-β-cyclodextrin. Food Funct 2021;12:5018-26. [Crossref] [PubMed]
79. Rosa F, Sharma AK, Gurung M, et al. Human Milk Oligosaccharides Impact Cellular and Inflammatory Gene Expression and Immune Response. Front Immunol 2022;13:907529. [Crossref] [PubMed]
80. Autran CA, Kellman BP, Kim JH, et al. Human milk oligosaccharide composition predicts risk of necrotising enterocolitis in preterm infants. Gut 2018;67:1064-70. [Crossref] [PubMed]
81. Huang W, Cho KY, Meng D, et al. The impact of indole-3-lactic acid on immature intestinal innate immunity and development: a transcriptomic analysis. Sci Rep 2021;11:8088. [Crossref] [PubMed]
82. Huang S, Gao Y, Wang Z, et al. Anti-inflammatory actions of acetate, propionate, and butyrate in fetal mouse jejunum cultures ex vivo and immature small intestinal cells in vitro. Food Sci Nutr 2022;10:564-76. [Crossref] [PubMed]
83. Gao Y, Davis B, Zhu W, et al. Short-chain fatty acid butyrate, a breast milk metabolite, enhances immature intestinal barrier function genes in response to inflammation in vitro and in vivo. Am J Physiol Gastrointest Liver Physiol 2021;320:G521-30. [Crossref] [PubMed]
84. He Y, Du W, Xiao S, et al. Colonization of fecal microbiota from patients with neonatal necrotizing enterocolitis exacerbates intestinal injury in germfree mice subjected to necrotizing enterocolitis-induction protocol via alterations in butyrate and regulatory T cells. J Transl Med 2021;19:510. [Crossref] [PubMed]
85. Yu W, Venkatraman A, Menden HL, et al. Short-chain fatty acids ameliorate necrotizing enterocolitis-like intestinal injury through enhancing Notch1-mediated single immunoglobulin interleukin-1-related receptor, toll-interacting protein, and A20 induction. Am J Physiol Gastrointest Liver Physiol 2023;324:G24-37. [Crossref] [PubMed]
86. Zheng N, Gao Y, Zhu W, et al. Short chain fatty acids produced by colonizing intestinal commensal bacterial interaction with expressed breast milk are anti-inflammatory in human immature enterocytes. PLoS One 2020;15:e0229283. [Crossref] [PubMed]
87. Lu P, Yamaguchi Y, Fulton WB, et al. Maternal aryl hydrocarbon receptor activation protects newborns against necrotizing enterocolitis. Nat Commun 2021;12:1042. [Crossref] [PubMed]
88. Nolan LS, Mihi B, Agrawal P, et al. Indole-3-Carbinol-Dependent Aryl Hydrocarbon Receptor Signaling Attenuates the Inflammatory Response in Experimental Necrotizing Enterocolitis. Immunohorizons 2021;5:193-209. [Crossref] [PubMed]
89. Larke JA, Kuhn-Riordon K, Taft DH, et al. Preterm Infant Fecal Microbiota and Metabolite Profiles Are Modulated in a Probiotic Specific Manner. J Pediatr Gastroenterol Nutr 2022;75:535-42. [Crossref] [PubMed]
90. Yin C, Fan P, Mou X, et al. Clostridium butyricum Ameliorates Atherosclerosis by Regulating Host Linoleic Acid Metabolism. Microorganisms 2025;13:1220. [Crossref] [PubMed]
91. Jia L, Jiang Y, Wu L, et al. Porphyromonas gingivalis aggravates colitis via a gut microbiota-linoleic acid metabolism-Th17/Treg cell balance axis. Nat Commun 2024;15:1617. [Crossref] [PubMed]
92. Pammi M, Suresh G. Enteral lactoferrin supplementation for prevention of sepsis and necrotizing enterocolitis in preterm infants. Cochrane Database Syst Rev 2020;3:CD007137. [Crossref] [PubMed]
93. Wang J, He Y, Liu Z, et al. Glutamine Peptides: Preparation, Analysis, Applications, and Their Role in Intestinal Barrier Protection. Nutrients 2025;17:1017. [Crossref] [PubMed]
94. Alshaikh BN, Reyes Loredo A, Yusuf K, et al. Enteral long-chain polyunsaturated fatty acids and necrotizing enterocolitis: A systematic review and meta-analysis. Am J Clin Nutr 2023;117:918-29. [Crossref] [PubMed]
95. Buddington RK, Davis SL, Buddington KK. The Risk of Necrotizing Enterocolitis Differs Among Preterm Pigs Fed Formulas With Either Lactose or Maltodextrin. J Pediatr Gastroenterol Nutr 2018;66:e61-6. [Crossref] [PubMed]
96. Call L, Stoll B, Oosterloo B, et al. Metabolomic signatures distinguish the impact of formula carbohydrates on disease outcome in a preterm piglet model of NEC. Microbiome 2018;6:111. [Crossref] [PubMed]
97. Pammi M, Cope J, Tarr PI, et al. Intestinal dysbiosis in preterm infants preceding necrotizing enterocolitis: a systematic review and meta-analysis. Microbiome 2017;5:31. [Crossref] [PubMed]
98. Warner BB, Deych E, Zhou Y, et al. Gut bacteria dysbiosis and necrotising enterocolitis in very low birthweight infants: a prospective case-control study. Lancet 2016;387:1928-36. [Crossref] [PubMed]
99. Stewart CJ, Embleton ND, Marrs EC, et al. Temporal bacterial and metabolic development of the preterm gut reveals specific signatures in health and disease. Microbiome 2016;4:67. [Crossref] [PubMed]
100. Tennoune N, Andriamihaja M, Blachier F. Production of Indole and Indole-Related Compounds by the Intestinal Microbiota and Consequences for the Host: The Good, the Bad, and the Ugly. Microorganisms 2022;10:930. [Crossref] [PubMed]
101. Tarracchini C, Milani C, Longhi G, et al. Unraveling the Microbiome of Necrotizing Enterocolitis: Insights in Novel Microbial and Metabolomic Biomarkers. Microbiol Spectr 2021;9:e0117621. [Crossref] [PubMed]
102. Fu X, Li S, Jiang Y, et al. Necrotizing Enterocolitis and Intestinal Microbiota: The Timing of Disease and Combined Effects of Multiple Species. Front Pediatr 2021;9:657349. [Crossref] [PubMed]