Gut microbiome and necrotizing enterocolitis: Understanding the connection to find a cure

Necrotizing enterocolitis (NEC), a cause of death among premature babies, has defied therapeutics for decades. Bacterial analyses have expanded insights into NEC pathophysiology and roles of the gut microbiome. We discuss the contribution of the gut microbiome and potential therapeutics, notably lactadherin, that may promote gut homeostasis to alleviate NEC. Necrotizing enterocolitis (NEC), a cause of death among premature babies, has defied therapeutics for decades. Bacterial analyses have expanded insights into NEC pathophysiology and roles of the gut microbiome. We discuss the contribution of the gut microbiome and potential therapeutics, notably lactadherin, that may promote gut homeostasis to alleviate NEC. Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease of prematurity. Its prevalence is about 7% among infants born at <32 weeks’ gestation with a birth weight of <1,500 g in the US (Neu and Walker, 2011Neu J. Walker W.A. Necrotizing enterocolitis.N. Engl. J. Med. 2011; 364: 255-264https://doi.org/10.1056/nejmra1005408Crossref PubMed Scopus (0) Google Scholar). Tragically, the rate of death resulting from complications of NEC, such as late-onset sepsis, is about 30% (Neu and Walker, 2011Neu J. Walker W.A. Necrotizing enterocolitis.N. Engl. J. Med. 2011; 364: 255-264https://doi.org/10.1056/nejmra1005408Crossref PubMed Scopus (0) Google Scholar). The gut microbiome, and particularly its composition at this premature state, has been linked to NEC. Under healthy conditions, the microbiome represents a high diversity of bacteria in balance, supporting metabolism alongside host defense against pathogens (Vangay et al., 2015Vangay P. Ward T. Gerber J.S. Knights D. Antibiotics, pediatric dysbiosis, and disease.Cell Host Microbe. 2015; 17: 553-564https://doi.org/10.1016/j.chom.2015.04.006Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar). Bacteria out of balance, dysbiosis, fail to perform these functions. Although the consensus is that the intrauterine environment during a healthy pregnancy is presumed to be free of bacteria, evidence suggests that the fetus develops with limited exposure to the microbiome until birth (Mueller et al., 2015Mueller N.T. Bakacs E. Combellick J. Grigoryan Z. Dominguez-Bello M.G. The infant microbiome development: mom matters.Trends Mol. Med. 2015; 21: 109-117https://doi.org/10.1016/j.molmed.2014.12.002Abstract Full Text Full Text PDF PubMed Scopus (594) Google Scholar), when rapid bacterial colonization occurs. Several factors—such as the mode of delivery (vaginal versus cesarean), feeding, medications, and transfusion—impact commensal colonization and lead to divergent and abnormal intestinal microbiota composition in premature newborns compared to full-term infants (Vangay et al., 2015Vangay P. Ward T. Gerber J.S. Knights D. Antibiotics, pediatric dysbiosis, and disease.Cell Host Microbe. 2015; 17: 553-564https://doi.org/10.1016/j.chom.2015.04.006Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar). Both commensal and pathogenic bacteria contain microbial-associated molecular patterns (MAMPs) that recognize the pattern recognition receptors (PRRs) on the intestinal mucosa (Denning and Prince, 2018Denning N.L. Prince J.M. Neonatal intestinal dysbiosis in necrotizing enterocolitis.Mol. Med. 2018; 24: 4https://doi.org/10.1186/s10020-018-0002-0Crossref PubMed Scopus (64) Google Scholar). The gut microbiome composition is highly relevant, as certain MAMPs present on specific bacteria may activate inflammatory pathways, disrupt the homeostasis, and influence NEC risk (Denning and Prince, 2018Denning N.L. Prince J.M. Neonatal intestinal dysbiosis in necrotizing enterocolitis.Mol. Med. 2018; 24: 4https://doi.org/10.1186/s10020-018-0002-0Crossref PubMed Scopus (64) Google Scholar). The intestinal microbiome typically displays a commensal relationship with the host. Alterations of the intestinal microbiome (dysbiosis) lead preterm infants to develop NEC (Vangay et al., 2015Vangay P. Ward T. Gerber J.S. Knights D. Antibiotics, pediatric dysbiosis, and disease.Cell Host Microbe. 2015; 17: 553-564https://doi.org/10.1016/j.chom.2015.04.006Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar). Molecular profiling of the microbiota, through sequencing of the highly conserved 16S small subunit bacterial ribosomal ribonucleic acid (rRNA) genes or metagenomic analysis, allows high-throughput screening of previously unidentified microbes, enhancing our understanding of the microbiota that predisposes to NEC. The composition of the gut microbiome is dynamic and influenced by the environment, and it changes from infancy (Vangay et al., 2015Vangay P. Ward T. Gerber J.S. Knights D. Antibiotics, pediatric dysbiosis, and disease.Cell Host Microbe. 2015; 17: 553-564https://doi.org/10.1016/j.chom.2015.04.006Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar). Traditionally, infants were believed to have a sterile gut that bacteria begin to populate only after delivery, since early testing methods failed to detect bacteria from the amniotic fluid or the surface of newborns after delivery with culture techniques. Using 16S rRNA gene sequencing, Firmicutes, Tenericutes, Proteobacteria, Bacteroides, and Fusobacteria communities were found on the placental specimen (Denning and Prince, 2018Denning N.L. Prince J.M. Neonatal intestinal dysbiosis in necrotizing enterocolitis.Mol. Med. 2018; 24: 4https://doi.org/10.1186/s10020-018-0002-0Crossref PubMed Scopus (64) Google Scholar). In addition, several studies revealed the presence of bacteria within the umbilical cord and meconium of infants before initiation of feedings (Denning and Prince, 2018Denning N.L. Prince J.M. Neonatal intestinal dysbiosis in necrotizing enterocolitis.Mol. Med. 2018; 24: 4https://doi.org/10.1186/s10020-018-0002-0Crossref PubMed Scopus (64) Google Scholar). It is, therefore, possible that the gut begins to colonize with bacteria in utero. Over time, the community becomes expanded and more complex so that by adulthood, the human intestinal microbiome harbors nearly 100 trillion microbial species. Microbiota in healthy term infants is established in a stepwise process, starting with facultative anaerobes. Within a few weeks of life, Bifidobacterium species become prevalent. The diversity of the infant’s gut increases with time and undergoes a significant shift when a baby moves from breast milk to other diets (Denning and Prince, 2018Denning N.L. Prince J.M. Neonatal intestinal dysbiosis in necrotizing enterocolitis.Mol. Med. 2018; 24: 4https://doi.org/10.1186/s10020-018-0002-0Crossref PubMed Scopus (64) Google Scholar; Vangay et al., 2015Vangay P. Ward T. Gerber J.S. Knights D. Antibiotics, pediatric dysbiosis, and disease.Cell Host Microbe. 2015; 17: 553-564https://doi.org/10.1016/j.chom.2015.04.006Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar). In contrast, preterm infants are exposed to prenatal and postnatal environmental insults while the microbiome is still developing with low diversity. The prenatal microbiome is influenced by the neonatal intensive care unit environment, and community shifts may increase NEC risk. A decrease in species diversity and a predominance of proteobacteria have been demonstrated weeks before the development of the NEC (Denning and Prince, 2018Denning N.L. Prince J.M. Neonatal intestinal dysbiosis in necrotizing enterocolitis.Mol. Med. 2018; 24: 4https://doi.org/10.1186/s10020-018-0002-0Crossref PubMed Scopus (64) Google Scholar; Vangay et al., 2015Vangay P. Ward T. Gerber J.S. Knights D. Antibiotics, pediatric dysbiosis, and disease.Cell Host Microbe. 2015; 17: 553-564https://doi.org/10.1016/j.chom.2015.04.006Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar). The impact of dysbiosis in NEC was explored in a study involving the microbiomes of twins, with only one of them developing NEC (Hourigan et al., 2016Hourigan S.K. Ta A. Wong W.S. Clemency N.C. Provenzano M.G. Baveja R. Iyer R. Klein E. Niederhuber J.E. The microbiome in necrotizing enterocolitis: A case report in twins and minireview.Clin. Ther. 2016; 38: 747-753https://doi.org/10.1016/j.clinthera.2016.02.014Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). 16S rRNA sequencing of their stool samples revealed that the infant that developed NEC initially had a diverse microbiome, but this diversity was abruptly lost weeks before the development of NEC. Microbiome imbalance was caused by colonization with a major phylum of a Gram-negative bacterium. The baby without NEC had a prominence of Firmicutes in all samples collected over time, whereas the baby with NEC had both Proteobacteria and Firmicutes, with an increasing amount of Proteobacteria weeks before developing NEC (Hourigan et al., 2016Hourigan S.K. Ta A. Wong W.S. Clemency N.C. Provenzano M.G. Baveja R. Iyer R. Klein E. Niederhuber J.E. The microbiome in necrotizing enterocolitis: A case report in twins and minireview.Clin. Ther. 2016; 38: 747-753https://doi.org/10.1016/j.clinthera.2016.02.014Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). Even the intestinal microbiome of healthy premature infants without NEC differs from that of healthy full-term infants. Samples from full-term infants were found to be clustered with similar bacteria at all time-points of the first eight weeks of life. In contrast, healthy premature infants revealed clustering at unique intervals, suggesting that the time course of the development of the microbiome is important in premature infants (Hourigan et al., 2016Hourigan S.K. Ta A. Wong W.S. Clemency N.C. Provenzano M.G. Baveja R. Iyer R. Klein E. Niederhuber J.E. The microbiome in necrotizing enterocolitis: A case report in twins and minireview.Clin. Ther. 2016; 38: 747-753https://doi.org/10.1016/j.clinthera.2016.02.014Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). The pattern of evolutionary change in bacteria may create an unbalanced microbiome with both increased pathogens and decreased protective species, leading to susceptibility to NEC. Thus, active microbiome balancing may provide a novel strategy for preventing NEC. Given the role of dysbiosis in NEC, understanding the microbiome function is crucial. Preterm infant dysbiosis may contribute to an immature barrier function and maladaptive immune responses, increasing the risk of NEC (Niño et al., 2016Niño D.F. Sodhi C.P. Hackam D.J. Necrotizing enterocolitis: new insights into pathogenesis and mechanisms.Nat. Rev. Gastroenterol. Hepatol. 2016; 13: 590-600https://doi.org/10.1038/nrgastro.2016.119Crossref PubMed Scopus (347) Google Scholar). From this perspective, the three key functions of the microbiome are metabolism, training and development of the immune system, and protection against disease. Without the contribution of the gut microbiome, human metabolic pathways would lack several nutrients, vitamins, and amino acids. In addition, the gut microbiome helps ferment non-digestible fibers that produce short-chain fatty acids (SCFA), i.e., acetate, propionate, and butyrate (Niño et al., 2016Niño D.F. Sodhi C.P. Hackam D.J. Necrotizing enterocolitis: new insights into pathogenesis and mechanisms.Nat. Rev. Gastroenterol. Hepatol. 2016; 13: 590-600https://doi.org/10.1038/nrgastro.2016.119Crossref PubMed Scopus (347) Google Scholar). Butyrate is an important energy source for the cells that constitute the epithelium of the human colon. Hence, SCFA deficiency causes leaky gut, bacterial translocation, and increased local inflammation. The gut commensals help in mucosal barrier function (Niño et al., 2016Niño D.F. Sodhi C.P. Hackam D.J. Necrotizing enterocolitis: new insights into pathogenesis and mechanisms.Nat. Rev. Gastroenterol. Hepatol. 2016; 13: 590-600https://doi.org/10.1038/nrgastro.2016.119Crossref PubMed Scopus (347) Google Scholar). The epithelium produces mucus and anti-microbial molecules to inhibit pathogen invasion (Kamada et al., 2013Kamada N. Seo S.U. Chen G.Y. Núñez G. Role of the gut microbiota in immunity and inflammatory disease.Nat. Rev. Immunol. 2013; 13: 321-335https://doi.org/10.1038/nri3430Crossref PubMed Scopus (1511) Google Scholar). The observation that the mucus layer in germ-free mice is thinner than in conventional mice points to the contribution of the microbiota to mucus production. Interestingly, oral administration of the bacterial products restores the mucus layer thickness of germ-free mice to normal (Niño et al., 2016Niño D.F. Sodhi C.P. Hackam D.J. Necrotizing enterocolitis: new insights into pathogenesis and mechanisms.Nat. Rev. Gastroenterol. Hepatol. 2016; 13: 590-600https://doi.org/10.1038/nrgastro.2016.119Crossref PubMed Scopus (347) Google Scholar). The gut microbiota is essential for the development and functional maturation of the gut immune system, including gut-associated lymphoid tissue, regulatory T-cells, IgA-producing B-cells, and innate lymphoid cells (ILCs) (Niño et al., 2016Niño D.F. Sodhi C.P. Hackam D.J. Necrotizing enterocolitis: new insights into pathogenesis and mechanisms.Nat. Rev. Gastroenterol. Hepatol. 2016; 13: 590-600https://doi.org/10.1038/nrgastro.2016.119Crossref PubMed Scopus (347) Google Scholar; Kamada et al., 2013Kamada N. Seo S.U. Chen G.Y. Núñez G. Role of the gut microbiota in immunity and inflammatory disease.Nat. Rev. Immunol. 2013; 13: 321-335https://doi.org/10.1038/nri3430Crossref PubMed Scopus (1511) Google Scholar). In addition, the gut microbiota prevents exogenous pathogen infection through direct competition for common nutrients and niches, resulting in indirect enhancement of host defense mechanisms. Under homeostasis, lamina propria macrophages and dendritic cells (DCs, gut resident phagocytes) exhibit unresponsiveness to commensal bacteria. Gut commensal microbiota is essential for upregulating the production of pro-IL-1β, the precursor to IL-1β, in resident phagocytes. But, they cannot induce the processing of pro-IL-1β into biologically active mature IL-1β, and thus a state of hyporesponsiveness is maintained (Kamada et al., 2013Kamada N. Seo S.U. Chen G.Y. Núñez G. Role of the gut microbiota in immunity and inflammatory disease.Nat. Rev. Immunol. 2013; 13: 321-335https://doi.org/10.1038/nri3430Crossref PubMed Scopus (1511) Google Scholar). Upon infection by enteric pathogens, such as Salmonella typhimurium, Pseudomonas aeruginosa can induce the processing of pro-IL-1β by promoting the activation of caspase-1 via the inflammasome pathway. The protective role of IL-1β in intestinal immunity is partly mediated by its ability to induce the expression of endothelial adhesion molecules that contribute to neutrophil recruitment and pathogen clearance in the intestine (Kamada et al., 2013Kamada N. Seo S.U. Chen G.Y. Núñez G. Role of the gut microbiota in immunity and inflammatory disease.Nat. Rev. Immunol. 2013; 13: 321-335https://doi.org/10.1038/nri3430Crossref PubMed Scopus (1511) Google Scholar; Niño et al., 2016Niño D.F. Sodhi C.P. Hackam D.J. Necrotizing enterocolitis: new insights into pathogenesis and mechanisms.Nat. Rev. Gastroenterol. Hepatol. 2016; 13: 590-600https://doi.org/10.1038/nrgastro.2016.119Crossref PubMed Scopus (347) Google Scholar). The intestinal microbiota also promotes T-cell differentiation and immunity by producing IL-22 by ILCs (Niño et al., 2016Niño D.F. Sodhi C.P. Hackam D.J. Necrotizing enterocolitis: new insights into pathogenesis and mechanisms.Nat. Rev. Gastroenterol. Hepatol. 2016; 13: 590-600https://doi.org/10.1038/nrgastro.2016.119Crossref PubMed Scopus (347) Google Scholar; Kamada et al., 2013Kamada N. Seo S.U. Chen G.Y. Núñez G. Role of the gut microbiota in immunity and inflammatory disease.Nat. Rev. Immunol. 2013; 13: 321-335https://doi.org/10.1038/nri3430Crossref PubMed Scopus (1511) Google Scholar). Utilizing an NEC model in 7-day-old mice reveals that binding bacteria by maternally derived IgA promotes diversity in the microbiome during the critical window when infants lack IgA. Shaping the neonate’s gut microbiota with IgA leads to decreased Enterobacteriaceae-induced inflammation and normal gut development (Gopalakrishna et al., 2019Gopalakrishna K.P. Macadangdang B.R. Rogers M.B. Tometich J.T. Firek B.A. Baker R. Ji J. Burr A.H.P. Ma C. Good M. et al.Maternal IgA protects against the development of necrotizing enterocolitis in preterm infants.Nat. Med. 2019; 25: 1110-1115https://doi.org/10.1038/s41591-019-0480-9Crossref PubMed Scopus (151) Google Scholar). The intestinal immaturity of the premature gut is likely a key causative factor in NEC. When exposed to the luminal microbiota of premature infants, an underdeveloped gut immune system leads to uncontrolled inflammation (Niño et al., 2016Niño D.F. Sodhi C.P. Hackam D.J. Necrotizing enterocolitis: new insights into pathogenesis and mechanisms.Nat. Rev. Gastroenterol. Hepatol. 2016; 13: 590-600https://doi.org/10.1038/nrgastro.2016.119Crossref PubMed Scopus (347) Google Scholar). Pathogenic and commensal bacteria penetrate the mucus layer to sense the epithelial cells’ PRRs and initiate an immune response. The most common PRR in the intestine is TLR4, expressed by epithelial cells, macrophages, and DCs, which detects MAMPs such as lipopolysaccharide (LPS). Evidence suggests that TLR4 is expressed at higher levels in the preterm intestine than the term infant (Niño et al., 2016Niño D.F. Sodhi C.P. Hackam D.J. Necrotizing enterocolitis: new insights into pathogenesis and mechanisms.Nat. Rev. Gastroenterol. Hepatol. 2016; 13: 590-600https://doi.org/10.1038/nrgastro.2016.119Crossref PubMed Scopus (347) Google Scholar). Activation of TLR4 on the lining of the premature intestine by Gram-negative bacteria that colonize the premature gut leads to increased cytokine release, enterocyte apoptosis, and impaired mucosal healing, which collectively drive NEC (Niño et al., 2016Niño D.F. Sodhi C.P. Hackam D.J. Necrotizing enterocolitis: new insights into pathogenesis and mechanisms.Nat. Rev. Gastroenterol. Hepatol. 2016; 13: 590-600https://doi.org/10.1038/nrgastro.2016.119Crossref PubMed Scopus (347) Google Scholar). Simultaneously, the translocation of bacteria through the gut mucosa activates TLR4 on the lining of the endothelium of the premature bowel mesentery, resulting in bowel necrosis and the release of damage-associated molecular patterns (DAMPs), further aggravating inflammation and precipitating septic shock (Niño et al., 2016Niño D.F. Sodhi C.P. Hackam D.J. Necrotizing enterocolitis: new insights into pathogenesis and mechanisms.Nat. Rev. Gastroenterol. Hepatol. 2016; 13: 590-600https://doi.org/10.1038/nrgastro.2016.119Crossref PubMed Scopus (347) Google Scholar; Kamada et al., 2013Kamada N. Seo S.U. Chen G.Y. Núñez G. Role of the gut microbiota in immunity and inflammatory disease.Nat. Rev. Immunol. 2013; 13: 321-335https://doi.org/10.1038/nri3430Crossref PubMed Scopus (1511) Google Scholar). The loss of enteric glia causes intestinal dysmotility as manifested in NEC patients. TLR4 on enteric glia causes depletion of glia, as demonstrated in a recent study where mice lacking TLR4 on enteric glia did not show NEC-induced enteric glia loss and were protected from NEC (Kovler et al., 2021Kovler M.L. Gonzalez Salazar A.J. Fulton W.B. Lu P. Yamaguchi Y. Zhou Q. Sampah M. Ishiyama A. Prindle Jr., T. Wang S. et al.Toll-like receptor 4-mediated enteric glia loss is critical for the development of necrotizing enterocolitis.Sci. Transl Med. 2021; 13: eabg3459https://doi.org/10.1126/scitranslmed.abg3459Crossref PubMed Scopus (23) Google Scholar). Interestingly, a brain-derived neurotrophic factor released from enteric glia restrained TLR4 signaling on the intestine to prevent NEC (Kovler et al., 2021Kovler M.L. Gonzalez Salazar A.J. Fulton W.B. Lu P. Yamaguchi Y. Zhou Q. Sampah M. Ishiyama A. Prindle Jr., T. Wang S. et al.Toll-like receptor 4-mediated enteric glia loss is critical for the development of necrotizing enterocolitis.Sci. Transl Med. 2021; 13: eabg3459https://doi.org/10.1126/scitranslmed.abg3459Crossref PubMed Scopus (23) Google Scholar). TLR4 is expressed at high levels in the developing gut in utero and then decreases after birth. The elevated basal level of TLR4 supports in utero gut development through Notch signaling pathway activation, which results in the survival and proliferation of intestinal stem cells (Niño et al., 2016Niño D.F. Sodhi C.P. Hackam D.J. Necrotizing enterocolitis: new insights into pathogenesis and mechanisms.Nat. Rev. Gastroenterol. Hepatol. 2016; 13: 590-600https://doi.org/10.1038/nrgastro.2016.119Crossref PubMed Scopus (347) Google Scholar). In the setting of premature birth, intestinal TLR4 levels remain elevated because gut development has not been completed and also because of the early presence and consecutive activation by luminal microorganisms. Upon colonization of the gut by bacteria in the postnatal period, in utero TLR4 signaling required for healthy gut development transforms into a destructive phenomenon in the postnatal period (Niño et al., 2016Niño D.F. Sodhi C.P. Hackam D.J. Necrotizing enterocolitis: new insights into pathogenesis and mechanisms.Nat. Rev. Gastroenterol. Hepatol. 2016; 13: 590-600https://doi.org/10.1038/nrgastro.2016.119Crossref PubMed Scopus (347) Google Scholar). Breast milk contains bioactive factors that positively impact host immunity, inflammation, and mucosal protection. Additionally, breastfed infants are less likely to develop NEC than formula-fed infants (Asbury et al., 2020Asbury M.R. Butcher J. Copeland J.K. Unger S. Bando N. Comelli E.M. Forte V. Kiss A. LeMay-Nedjelski L. Sherman P.M. et al.Mothers of preterm infants have individualized breast milk microbiota that changes temporally based on maternal characteristics.Cell Host Microbe. 2020; 28 (e664): 669-682.e4https://doi.org/10.1016/j.chom.2020.08.001Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Breastmilk notably increases the diversity of gut bacterial colonization (Pannaraj et al., 2017Pannaraj P.S. Li F. Cerini C. Bender J.M. Yang S. Rollie A. Adisetiyo H. Zabih S. Lincez P.J. Bittinger K. et al.Association between breast milk bacterial communities and establishment and development of the infant gut microbiome.JAMA Pediatr. 2017; 171: 647-654https://doi.org/10.1001/jamapediatrics.2017.0378Crossref PubMed Scopus (617) Google Scholar). It has immunomodulatory factors such as secretory IgA, mucin, lysozyme, anti-inflammatory cytokines, lactoferrin, growth factors, enzymes, oligosaccharides, polyunsaturated fatty acids, L-arginine, glutamine, prebiotics that formula lacks. These factors induce mucosal protection and inhibit inflammation (Pannaraj et al., 2017Pannaraj P.S. Li F. Cerini C. Bender J.M. Yang S. Rollie A. Adisetiyo H. Zabih S. Lincez P.J. Bittinger K. et al.Association between breast milk bacterial communities and establishment and development of the infant gut microbiome.JAMA Pediatr. 2017; 171: 647-654https://doi.org/10.1001/jamapediatrics.2017.0378Crossref PubMed Scopus (617) Google Scholar). Maternal IgA is a critical factor in breastmilk for NEC prevention because it shapes the host-microbiota relationship of preterm neonates. IgA binding to the intestinal bacteria of preterm infants is positively linked to breastfeeding and negatively correlated with NEC development (Gopalakrishna et al., 2019Gopalakrishna K.P. Macadangdang B.R. Rogers M.B. Tometich J.T. Firek B.A. Baker R. Ji J. Burr A.H.P. Ma C. Good M. et al.Maternal IgA protects against the development of necrotizing enterocolitis in preterm infants.Nat. Med. 2019; 25: 1110-1115https://doi.org/10.1038/s41591-019-0480-9Crossref PubMed Scopus (151) Google Scholar). Glutamine and nucleotides may help in gastrointestinal cell metabolism (Pannaraj et al., 2017Pannaraj P.S. Li F. Cerini C. Bender J.M. Yang S. Rollie A. Adisetiyo H. Zabih S. Lincez P.J. Bittinger K. et al.Association between breast milk bacterial communities and establishment and development of the infant gut microbiome.JAMA Pediatr. 2017; 171: 647-654https://doi.org/10.1001/jamapediatrics.2017.0378Crossref PubMed Scopus (617) Google Scholar; Asbury et al., 2020Asbury M.R. Butcher J. Copeland J.K. Unger S. Bando N. Comelli E.M. Forte V. Kiss A. LeMay-Nedjelski L. Sherman P.M. et al.Mothers of preterm infants have individualized breast milk microbiota that changes temporally based on maternal characteristics.Cell Host Microbe. 2020; 28 (e664): 669-682.e4https://doi.org/10.1016/j.chom.2020.08.001Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Epidermal growth factor (EGF) can directly improve gastrointestinal function and promote gut maturity. Evidence shows that breastmilk inhibits TLR4 signaling by preventing glycogen synthase kinase 3β activity (Good et al., 2015Good M. Sodhi C.P. Egan C.E. Afrazi A. Jia H. Yamaguchi Y. Lu P. Branca M.F. Ma C. Prindle Jr., T. et al.Breast milk protects against the development of necrotizing enterocolitis through inhibition of Toll-like receptor 4 in the intestinal epithelium via activation of the epidermal growth factor receptor.Mucosal Immunol. 2015; 8: 1166-1179https://doi.org/10.1038/mi.2015.30Crossref PubMed Scopus (163) Google Scholar), suggesting the presence of obscured TLR4 antagonists in milk or, presumably, the above factors having an anti-TLR4 function. Moreover, the beneficial effects of breastmilk are partially dependent upon activation of EGF receptor signaling (Good et al., 2015Good M. Sodhi C.P. Egan C.E. Afrazi A. Jia H. Yamaguchi Y. Lu P. Branca M.F. Ma C. Prindle Jr., T. et al.Breast milk protects against the development of necrotizing enterocolitis through inhibition of Toll-like receptor 4 in the intestinal epithelium via activation of the epidermal growth factor receptor.Mucosal Immunol. 2015; 8: 1166-1179https://doi.org/10.1038/mi.2015.30Crossref PubMed Scopus (163) Google Scholar). Whether the development of NEC in association with formula feeding represents the presence of harmful components in infant formula or the deficiency of a protective agent that is only present in breastmilk remains to be determined. Breast milk contains milk fat globule membrane (MFGM), composed of lipids with interspersed membrane-bound proteins, glycoproteins, enzymes, and cholesterol resulting in a bioactive molecule that confers protective effects of breast milk (Bhinder et al., 2017Bhinder G. Allaire J.M. Garcia C. Lau J.T. Chan J.M. Ryz N.R. Bosman E.S. Graef F.A. Crowley S.M. Celiberto L.S. et al.Milk fat globule membrane supplementation in formula modulates the neonatal gut microbiome and normalizes intestinal development.Sci. Rep. 2017; 7: 45274https://doi.org/10.1038/srep45274Crossref PubMed Scopus (113) Google Scholar). Most formulas lack MFGM. As such, MFGM supplementation in formula shapes neonatal gut microbiome balance (Bhinder et al., 2017Bhinder G. Allaire J.M. Garcia C. Lau J.T. Chan J.M. Ryz N.R. Bosman E.S. Graef F.A. Crowley S.M. Celiberto L.S. et al.Milk fat globule membrane supplementation in formula modulates the neonatal gut microbiome and normalizes intestinal development.Sci. Rep. 2017; 7: 45274https://doi.org/10.1038/srep45274Crossref PubMed Scopus (113) Google Scholar). Lactadherin, also named milk fat globule-EGF factor VIII, is a pivotal glycoprotein of MFGM. It is also produced by macrophages and epithelial cells and plays an anti-inflammatory role in sepsis, intestinal ischemia/reperfusion (I/R) injury, and inflammatory bowel disease in rodents (Aziz et al., 2017Aziz M. Hansen L.W. Prince J.M. Wang P. Chapter 2 - role of MFG-E8 in neonatal inflammation.in: Watson R.R. Collier R.J. Preedy V.R. Dairy in Human Health and Disease Across the Lifespan. Academic Press, 2017: 21-30Crossref Scopus (3) Google Scholar). Treatment with lactadherin ameliorates inflammation and organ injury in murine neonatal sepsis (Aziz et al., 2017Aziz M. Hansen L.W. Prince J.M. Wang P. Chapter 2 - role of MFG-E8 in neonatal inflammation.in: Watson R.R. Collier R.J. Preedy V.R. Dairy in Human Health and Disease Across the Lifespan. Academic Press, 2017: 21-30Crossref Scopus (3) Google Scholar). Clinically, decreased levels of lactadhrin are associated with NEC severity in preterm neonates (Asaro et al., 2021Asaro J.A. Khan Z. Brewer M. Klose K. Pesce C. Schanler R.J. Codipilly C.N. Relationship between milk fat globule-epidermal growth factor 8 and intestinal cytokines in infants born preterm.J. Pediatr. 2021; 230 (e71): 71-75.e1https://doi.org/10.1016/j.jpeds.2020.11.014Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar). Given the importance of lactadherin against inflammation, lactadherin may be a therapeutic candidate in NEC. Understanding the mechanisms of dysbiosis, causing NEC (devastation), and the pathway of protecting against NEC through lactadherin-mediated intestinal homeostasis (salvation) may reveal novel insights into the broad scope of NEC (Figure 1). Lactadherin serves as an opsonin for apoptotic cells and enhances the phagocytic clearance of senescent cells (Aziz et al., 2017Aziz M. Hansen L.W. Prince J.M. Wang P. Chapter 2 - role of MFG-E8 in neonatal inflammation.in: Watson R.R. Collier R.J. Preedy V.R. Dairy in Human Health and Disease Across the Lifespan. Academic Press, 2017: 21-30Crossref Scopus (3) Google Scholar). This function of lactadherin is implicated in preventing inflammatory and autoimmune diseases by reducing secondary necrosis, preventing spillover of DAMPs, and inducing the release of anti-inflammatory cytokines IL-10 and TGFβ for immunoregulation. In addition, lactadherin also generates a direct anti-inflammatory effect, utilizing the STAT3 pathway by downregulating the LPS-induced TNFα production in macrophages through activation of suppressor of cytokine signaling 3 (SOCS3). Lactadherin-induced SOCS3 may in turn target the NF-kB p65 component and act as a negative regulator of LPS-mediated TLR4 signaling via NF-kB for cytokine production (Aziz et al., 2017Aziz M. Hansen L.W. Prince J.M. Wang P. Chapter 2 - role of MFG-E8 in neonatal inflammation.in: Watson R.R. Collier R.J. Preedy V.R. Dairy in Human Health and Disease Across the Lifespan. Academic Press, 2017: 21-30Crossref Scopus (3) Google Scholar), the pathway associated with NEC pathophysiology. In many pro-inflammatory conditions—including sepsis, endotoxemia, intestinal injury, acute kidney injury, acute lung injury, colitis, and organ I/R models—lactadherin expression is downregulated. Specifically, in cecal ligation and puncture-induced sepsis, the expression of lactadherin decreases, leading to the accumulation of apoptotic cells, causing secondary necrosis and excessive inflammation accompanied by increased levels of TNFα, IL-6, IL-8, and myeloperoxidase (Aziz et al., 2017Aziz M. Hansen L.W. Prince J.M. Wang P. Chapter 2 - role of MFG-E8 in neonatal inflammation.in: Watson R.R. Collier R.J. Preedy V.R. Dairy in Human Health and Disease Across the Lifespan. Academic Press, 2017: 21-30Crossref Scopus (3) Google Scholar). Exogenous administration of lactadherin helps restore immune hemostasis by reducing inflammatory mediators and accelerating tissue regeneration. Lactadherin promotes the healing of injured intestinal mucosa by a PKC-dependent pathway. It also fosters vascularization via a vascular endothelial growth factor (VEGF)/protein kinase B (PKB) pathway (Aziz et al., 2017Aziz M. Hansen L.W. Prince J.M. Wang P. Chapter 2 - role of MFG-E8 in neonatal inflammation.in: Watson R.R. Collier R.J. Preedy V.R. Dairy in Human Health and Disease Across the Lifespan. Academic Press, 2017: 21-30Crossref Scopus (3) Google Scholar). In addition to M2 macrophage polarization, lactadherin promotes the development of intestinal DCs in the Peyer’s Patches by increasing the expression of signal regulatory protein and regulatory T- and B-cells and controls neutrophils’ exaggerated functions by producing more IL-10 and less IL-12 (Aziz et al., 2017Aziz M. Hansen L.W. Prince J.M. Wang P. Chapter 2 - role of MFG-E8 in neonatal inflammation.in: Watson R.R. Collier R.J. Preedy V.R. Dairy in Human Health and Disease Across the Lifespan. Academic Press, 2017: 21-30Crossref Scopus (3) Google Scholar). In this way, lactadherin plays an essential role in attenuating inflammation and improving prognosis in several animal models of life-threatening, inflammatory diseases. A growing body of evidence highlights the protective effect of MFGM, a membrane surrounding the milk’s lipid component milk fat globule, against infectious diseases, through modulation of the intestinal immune response and establishment of the early gut microbiota (Bhinder et al., 2017Bhinder G. Allaire J.M. Garcia C. Lau J.T. Chan J.M. Ryz N.R. Bosman E.S. Graef F.A. Crowley S.M. Celiberto L.S. et al.Milk fat globule membrane supplementation in formula modulates the neonatal gut microbiome and normalizes intestinal development.Sci. Rep. 2017; 7: 45274https://doi.org/10.1038/srep45274Crossref PubMed Scopus (113) Google Scholar; Asbury et al., 2020Asbury M.R. Butcher J. Copeland J.K. Unger S. Bando N. Comelli E.M. Forte V. Kiss A. LeMay-Nedjelski L. Sherman P.M. et al.Mothers of preterm infants have individualized breast milk microbiota that changes temporally based on maternal characteristics.Cell Host Microbe. 2020; 28 (e664): 669-682.e4https://doi.org/10.1016/j.chom.2020.08.001Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). MFGM contains glycoproteins and glycolipids, which have anti-microbial, anti-inflammatory, and prebiotic functions in the gut. The glycoprotein lactadherin of the MFGM interferes with pathogen attachment to the intestinal mucosa, causing the pathogens to be neutralized by the anti-microbial components of MFGM (Asbury et al., 2020Asbury M.R. Butcher J. Copeland J.K. Unger S. Bando N. Comelli E.M. Forte V. Kiss A. LeMay-Nedjelski L. Sherman P.M. et al.Mothers of preterm infants have individualized breast milk microbiota that changes temporally based on maternal characteristics.Cell Host Microbe. 2020; 28 (e664): 669-682.e4https://doi.org/10.1016/j.chom.2020.08.001Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar; Bhinder et al., 2017Bhinder G. Allaire J.M. Garcia C. Lau J.T. Chan J.M. Ryz N.R. Bosman E.S. Graef F.A. Crowley S.M. Celiberto L.S. et al.Milk fat globule membrane supplementation in formula modulates the neonatal gut microbiome and normalizes intestinal development.Sci. Rep. 2017; 7: 45274https://doi.org/10.1038/srep45274Crossref PubMed Scopus (113) Google Scholar). The glycoproteins of MFGM inhibit the intestinal adhesion of E. coli. and common enteric pathogens such as the Salmonella species and rotavirus (Bhinder et al., 2017Bhinder G. Allaire J.M. Garcia C. Lau J.T. Chan J.M. Ryz N.R. Bosman E.S. Graef F.A. Crowley S.M. Celiberto L.S. et al.Milk fat globule membrane supplementation in formula modulates the neonatal gut microbiome and normalizes intestinal development.Sci. Rep. 2017; 7: 45274https://doi.org/10.1038/srep45274Crossref PubMed Scopus (113) Google Scholar; Asbury et al., 2020Asbury M.R. Butcher J. Copeland J.K. Unger S. Bando N. Comelli E.M. Forte V. Kiss A. LeMay-Nedjelski L. Sherman P.M. et al.Mothers of preterm infants have individualized breast milk microbiota that changes temporally based on maternal characteristics.Cell Host Microbe. 2020; 28 (e664): 669-682.e4https://doi.org/10.1016/j.chom.2020.08.001Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Neonatal murine study to evaluate the role of lactadherin in sepsis prevention, a late complication of NEC, by suppressing intestinal inflammation and promoting cell survival identified a viable therapeutic strategy for lactadherin in critically ill infants with NEC (Aziz et al., 2017Aziz M. Hansen L.W. Prince J.M. Wang P. Chapter 2 - role of MFG-E8 in neonatal inflammation.in: Watson R.R. Collier R.J. Preedy V.R. Dairy in Human Health and Disease Across the Lifespan. Academic Press, 2017: 21-30Crossref Scopus (3) Google Scholar). Indeed, the lactadherin-deficient neonatal mice display favorable survival outcomes in sepsis (Aziz et al., 2017Aziz M. Hansen L.W. Prince J.M. Wang P. Chapter 2 - role of MFG-E8 in neonatal inflammation.in: Watson R.R. Collier R.J. Preedy V.R. Dairy in Human Health and Disease Across the Lifespan. Academic Press, 2017: 21-30Crossref Scopus (3) Google Scholar). In a prospective pilot study using healthy controls and NEC neonates, lactadherin was significantly downregulated in NEC neonates compared to healthy neonates (Asaro et al., 2021Asaro J.A. Khan Z. Brewer M. Klose K. Pesce C. Schanler R.J. Codipilly C.N. Relationship between milk fat globule-epidermal growth factor 8 and intestinal cytokines in infants born preterm.J. Pediatr. 2021; 230 (e71): 71-75.e1https://doi.org/10.1016/j.jpeds.2020.11.014Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar). Thus, lactadherin could be a possible biomarker related to the progression of NEC. Furthermore, in a rat model of NEC, which consists of aggressive gavage with formula, hypoxia, and cold stress, the NEC pups treated with recombinant human lactadherin showed significantly decreased pathological scores and incidence of NEC (Aziz et al., 2017Aziz M. Hansen L.W. Prince J.M. Wang P. Chapter 2 - role of MFG-E8 in neonatal inflammation.in: Watson R.R. Collier R.J. Preedy V.R. Dairy in Human Health and Disease Across the Lifespan. Academic Press, 2017: 21-30Crossref Scopus (3) Google Scholar). The improvement of neonatal NEC by treatment with lactadherin was mediated through the decreased expression of TLR4, reduced apoptosis of the intestinal epithelial cells, and maintenance of the integrity of the intestinal barrier (Aziz et al., 2017Aziz M. Hansen L.W. Prince J.M. Wang P. Chapter 2 - role of MFG-E8 in neonatal inflammation.in: Watson R.R. Collier R.J. Preedy V.R. Dairy in Human Health and Disease Across the Lifespan. Academic Press, 2017: 21-30Crossref Scopus (3) Google Scholar). Gut microbiome homeostasis prevents NEC in preterm neonates. Therefore, monitoring the microbiome development in premature infants helps identify preterms at higher risk of developing NEC. Breastfeeding is essential for protecting against NEC (Pannaraj et al., 2017Pannaraj P.S. Li F. Cerini C. Bender J.M. Yang S. Rollie A. Adisetiyo H. Zabih S. Lincez P.J. Bittinger K. et al.Association between breast milk bacterial communities and establishment and development of the infant gut microbiome.JAMA Pediatr. 2017; 171: 647-654https://doi.org/10.1001/jamapediatrics.2017.0378Crossref PubMed Scopus (617) Google Scholar; Good et al., 2015Good M. Sodhi C.P. Egan C.E. Afrazi A. Jia H. Yamaguchi Y. Lu P. Branca M.F. Ma C. Prindle Jr., T. et al.Breast milk protects against the development of necrotizing enterocolitis through inhibition of Toll-like receptor 4 in the intestinal epithelium via activation of the epidermal growth factor receptor.Mucosal Immunol. 2015; 8: 1166-1179https://doi.org/10.1038/mi.2015.30Crossref PubMed Scopus (163) Google Scholar). Individual milk components play a pivotal role in maintaining gut homeostasis and microbiome development in infants (Asbury et al., 2020Asbury M.R. Butcher J. Copeland J.K. Unger S. Bando N. Comelli E.M. Forte V. Kiss A. LeMay-Nedjelski L. Sherman P.M. et al.Mothers of preterm infants have individualized breast milk microbiota that changes temporally based on maternal characteristics.Cell Host Microbe. 2020; 28 (e664): 669-682.e4https://doi.org/10.1016/j.chom.2020.08.001Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar; Bhinder et al., 2017Bhinder G. Allaire J.M. Garcia C. Lau J.T. Chan J.M. Ryz N.R. Bosman E.S. Graef F.A. Crowley S.M. Celiberto L.S. et al.Milk fat globule membrane supplementation in formula modulates the neonatal gut microbiome and normalizes intestinal development.Sci. Rep. 2017; 7: 45274https://doi.org/10.1038/srep45274Crossref PubMed Scopus (113) Google Scholar). While the anti-inflammatory role of lactadherin in neonatal and adult inflammatory diseases perturbing gut homeostasis has been demonstrated, precise information regarding lactadherin’s role in the gut microbiome is lacking. Since lactadherin expression is downregulated in NEC infants (Asaro et al., 2021Asaro J.A. Khan Z. Brewer M. Klose K. Pesce C. Schanler R.J. Codipilly C.N. Relationship between milk fat globule-epidermal growth factor 8 and intestinal cytokines in infants born preterm.J. Pediatr. 2021; 230 (e71): 71-75.e1https://doi.org/10.1016/j.jpeds.2020.11.014Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar), it should be further investigated as a possible future therapeutic approach to reduce tissue injury and modulate the overactive immune system. Interestingly, an infant formula containing lactadherin is currently commercially available to improve gut health and microbial colonization. Whether genetic polymorphisms in lactadherin may account for variability in the risk of NEC needs to be determined. Similarly, epigenetics will be an exciting area in NEC pathophysiology to investigate. Studies manipulating lactadherin presence should be pursued to establish lactadherin’s role in shaping the gut microbiome. Subsequently, determining the mechanism of how lactadherin helps maintain the gut microbiome is crucial. Is it through the neutralization of the factors that alter the gut microbiome or directly by increasing the survival of the microbiomes affected in preterm infants? Translational studies on the effects of lactadherin or its probable analogs on gut microbiome development are essential. Addressing these future aims might identify lactadherin as a durable therapeutic that tips the microbiome balance to protect against NEC.