REvIEwS

Bench to bedside — new insights

into the pathogenesis of necrotizing
enterocolitis
David J. Hackam    ✉ and Chhinder P. Sodhi
Abstract | Necrotizing enterocolitis (NEC) is the leading cause of death and disability from
gastrointestinal disease in premature infants. Recent discoveries have shed light on a unifying
theorem to explain the pathogenesis of NEC, suggesting that specific treatments might finally be
forthcoming. A variety of experiments have highlighted how the interaction between bacterial
signalling receptors on the premature intestine and an abnormal gut microbiota incites a
pro-inflammatory response in the intestinal mucosa and its underlying endothelium that leads
to NEC. Central amongst the bacterial signalling receptors implicated in NEC development is
the lipopolysaccharide receptor Toll-​like receptor 4 (TLR4), which is expressed at higher levels
in the premature gut than in the full-​term gut. The high prenatal intestinal expression of TLR4
reflects the role of TLR4 in the regulation of normal gut development, and supports additional
studies indicating that NEC develops in response to signalling events that occur in utero.
This Review provides new evidence explaining the pathogenesis of NEC, explores new findings
indicating that NEC development has origins before birth, and discusses future questions and
opportunities for discovery in this field.

Necrotizing enterocolitis (NEC) is the leading cause of long-​term morbidity as the infant grows and develops7,8.
morbidity and mortality from gastrointestinal disease in Accumulating evidence in mice and piglets and vali-
premature infants. The worldwide incidence of NEC is dated in human tissue has identified a pivotal role for
7%, and a recent meta-​analysis including over 570,000 the bacterial receptor Toll-​like receptor 4 (TLR4) in trig-
infants revealed some reported geographical variation, gering an inflammatory response within the intestinal
which has been attributed to changes in incidence mucosa9,10, which leads to NEC after its interaction with
between high-​income and low-​income countries1,2. NEC the abnormal intestinal microbiota of the premature
is characterized by the acute onset of intestinal ischaemia host11–13. Additional studies have raised the intriguing
and necrosis, and carries a mortality of approximately possibility that the origins of NEC might exist before
25% in most cases, yet the mortality can be as high as birth, suggesting the opportunity for earlier, in utero
80% at 48 h after diagnosis in the most severe cases of interventions in NEC prevention14. Furthermore, the
fulminant NEC3. The high mortality associated with underlying processes that lead to the long-​term compli-
NEC reflects in part the lack of any specific treatment cations of NEC that affect the lung and brain have also
for patients with NEC4. NEC is currently managed by been partly elucidated and even reversed in preclinical
holding all feeds, decompressing the stomach with a models15–18, suggesting the potential for long-​term relief.
nasogastric tube, providing haemodynamic resuscitation Therefore, this Review is timed to assemble and interpret
through the administration of intravenous fluids and these latest findings, extend our insights into the mater-
inotropes, and provision of broad-​spectrum antibiotics nal origins of disease, and explore emerging therapeutics
that act against enteric microorganisms5. In patients who for NEC. We highlight how some of the older theories of
Division of Paediatric continue to exhibit clinical progression, urgent surgery is disease development — such as how NEC was thought to
Surgery, Johns Hopkins performed for resection of necrotic bowel and drainage arise as a simple consequence of an immature immune
University School of Medicine,
Baltimore, MD, USA.
of the abdominal cavity6. system19, or as a consequence of an immature intestinal
✉e-​mail: Dhackam1@ There is growing awareness that in addition to the barrier20, or as a specific lack of certain growth factors21
jhmi.edu detrimental effects of NEC on the intestine, the com- — can still be integrated within current thinking in the
https://doi.org/10.1038/ plications of NEC can also extend to secondary organs field, yet modified to reflect the latest advances. We also
s41575-022-00594-​x that include the lung and the brain, causing substantial discuss some of the most important open questions in

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lymphocytes34–36. These clinical and pathological features

Key points
of NEC inform our understanding of NEC pathogenesis,
• Necrotizing enterocolitis (NEC) is the leading cause of death from gastrointestinal as is discussed herein.
disease in premature infants and is characterized by the acute onset of patchy There is currently no specific treatment for NEC,
necrosis throughout the intestine, leading to systemic sepsis. and initial management involves cessation of feeds,
• NEC induction requires the activation of Toll-​like receptor 4 (TLR4) on the intestinal haemodynamic resuscitation and administration of
epithelium by the intestinal microbiota of the premature host, leading to enterocyte broad-​spectrum antibiotics37. In those patients who do
death, mucosal injury and translocation of bacteria into the circulation.
not respond to this first-​line treatment, abdominal sur-
• TLR4 is expressed at higher levels in the premature than in the full-​term intestine due gery is required, which typically involves removal of the
to its role in the regulation of normal gut development, and is inhibited by breast milk
dead and dying intestine38. Of the approximately 70%
and amniotic fluid in vitro and in vivo.
of patients who survive NEC32, there are two notable
• Studies in animals and human NEC tissue have shown that activation of the aryl additional comorbidities: neurocognitive impairment39
hydrocarbon receptor in utero can modulate the risk of NEC through effects on TLR4,
and lung disease18. Patients with NEC have a 2.6-​fold
potentially offering an opportunity during pregnancy for NEC prevention.
increased risk of neurocognitive impairment40, while
• Additional studies have identified causative roles for epigenetic modulation of key
severe NEC requiring surgery was seen in 25% of infants
signalling molecules and the inflammasome in NEC pathogenesis, and have shed light
on the effects of NEC on the developing brain and lung. with severe lung disease41. The overall global mortality
among infants with NEC has remained at approximately
• New prevention and therapeutic approaches for NEC are designed to interfere with
the abnormal host–microorganism signalling that occurs during the prenatal and
30% over many years40, with some variability between
early postnatal periods that lead to NEC. regions1, suggesting an urgent need to understand the
pathogenesis of NEC in greater detail42. Interestingly,
there are reports that mortality associated with NEC
the field that remain to be answered, including focus- might be substantially higher in low-​income and middle-
ing on bacteria-​induced host molecular pathways that income countries (LMICs) than in high-​income coun-
could lead to NEC development, the effects of meta­ tries, perhaps due to different gestational age cut-​offs for
bolism on the induction of NEC and how to predict resuscitation of extremely premature infants in LMICs,
NEC development. as has been suggested1.
Although this description reveals what NEC is, it is
Clinical considerations also critically important — for the purpose of describ-
The clinical presentation of NEC is unique among ing its underlying pathophysiology — to clearly identify
gastro­intestinal disorders. The disease usually begins what NEC is not. First, NEC is not simply inflammatory
in an otherwise stable infant in the neonatal intensive bowel disease (a diagnosis that refers to both ulcerative
care unit, nearly always after the administration of infant colitis and Crohn’s disease) of the premature newborn;
formula (as opposed to breast milk-​based) feeds, and neither ulcerative colitis nor Crohn’s disease is associ-
is typically initially characterized by abdominal disten- ated with the degree of intestinal necrosis, systemic sep-
tion and mild feeding intolerance22. These abdominal sis or the high mortality seen in NEC43. Furthermore,
symptoms can then progress to the acute development of the intestinal inflammation seen in infants who have
necrosis of the small and large intestine23 and are usually an intolerance to their formula could be confused with
followed by the development of overwhelming systemic NEC in the early phases, yet these infants generally
sepsis24. NEC affects between 1% and 12% of infants have a mild gastrointestinal disease that is usually self-
born before 37 weeks25,26. While all premature infants limiting 44. The haematochezia that is observed in
are at risk of NEC development, the risk is particularly patients with NEC must be distinguished from anal fis-
high in those under 1,500 g (refs27,28). The high incidence sures that can be readily excluded by physical examina-
of premature births across the globe, ranging from 5% of tion alone45. Likewise, NEC is very different from, but
all births in several European countries to 18% in some often confused with, a separate entity called spontaneous
African countries, reveals that patients are at risk of intestinal perforation (SIP), which is an acute process
NEC development worldwide29. Systematic reviews have that typically occurs within 3 to 9 days of life in infants
revealed a global increase in NEC incidence over time, under 1,500 g, in which a very small region of the small
with some studies showing an increase from 5% to 22% intestine spontaneously perforates in the absence of
among those with a birthweight <1,000 g over a 10-​year intestinal necrosis46. The confusion between NEC and
period30. Recent studies examining babies in the USA SIP lies in the fact that the initial symptoms (abdominal
have revealed that comprehensive risk reduction strat- distention and evidence of systemic sepsis) are similar
egies can reduce NEC incidence to below 2% of at-​risk between these entities47. However, the management and
infants31, although this finding has been challenged by outcome of these two diseases are very diferent39. NEC
studies from other regions, such as a recent cohort study should also be distinguished from intestinal dysmotil-
with data from the Spanish Neonatal Network revealing ity and ileus that result from an infection outside of the
that the incidence of NEC remained stable at 8.8% over abdomen, and whose inclusion in NEC datasets compli-
12 years (2005–2017)32. cates our understanding of the risk factors and patho-
Histologically, NEC is characterized by disruption logical underpinnings of NEC itself48. NEC must also be
of the intestinal epithelium and coagulative necrosis of distinguished from an unusual entity called pneumatosis
parts of the ileum and colon 33,34. These epithelial coli, a benign condition that affects mainly late preterm
changes are accompanied by a dense inflammatory or full-​term infants and is not associated with substantial
cell infiltrate that is particularly rich in monocytes and abdominal pain or distention49. These clinical findings

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point to the complex nature of NEC, its apparent overlap recognition receptor that is mutated in Crohn’s disease
with other neonatal diseases, and the need for a more and that recognizes molecules containing muramyl
refined molecular understanding of its pathogenesis. dipeptide (MDP)65, providing evidence for some patho-
genetic overlap between inflammatory bowel disease and
Pathogenesis NEC. The administration of the NOD2 agonist MDP to
A model for the pathogenesis of NEC. A unifying model mice attenuates NEC through secondary inhibition of
has emerged that explains the pathogenesis of NEC TLR4 (ref.64). Neal et al. found that this mucosal damage
and that is based on a critical role for the lipopolysac- facilitates the translocation of bacteria to the underlying
charide receptor TLR450,51 (Fig. 1). Initial proof for the microvasculature66, and Yazji et al. determined that the
importance of TLR4 signalling in NEC development is subsequent interaction between bacteria and TLR4 on
found in the observation that TLR4-​deficient mice are the endothelium of the mesentery leads to a reduction
protected from NEC9,52. By contrast, stop mutations in in the expression of endothelial nitric oxide synthase
the SIGIRR (single immunoglobulin IL-1-​related recep- (eNOS), vasoconstriction and the development of mes-
tor) gene, a critical inhibitor of TLR4 pathway53, were enteric ischaemia in mice67. In parallel, Egan et al. have
found in 10 of 17 human infants with severe NEC54. shown that the activation of TLR4 on the intestinal epi-
Sodhi et al. from our group determined that activation of thelium results in the recruitment of pro-​inflammatory
TLR4 by lipopolysaccharide on the intestinal epithelium T helper 17 (TH17) cells, which release pro-​inflammatory
as opposed to myeloid cells is critical for NEC develop- cytokines including IL-17 and IL-22 (ref.35), further driv-
ment, as mice lacking TLR4 on the intestinal epithelium ing the injury response in mice. More recently, Kovler
were protected from NEC11. Subsequent studies from et al. from our group showed that the TLR4-​mediated
our group showed that TLR4 signalling on the intesti- loss of enteric glia results in intestinal dysmotility and
nal epithelium of the premature host leads to enterocyte hyperinflammation in mice through a reduction in the
death by a combination of apoptosis55–57, autophagy21,58,59 release of the glial peptide brain-​derived neurotrophic
and necroptosis60, as well as impaired restitution61,62 and factor (BDNF), and the key findings were confirmed
reduced proliferation12,63, which together cause irrevers- in piglet models and in human tissue obtained from
ible damage to the intestinal mucosa. Richardson et al. infants undergoing surgery for NEC68. The observation
showed that TLR4 activation in the gut leading to NEC that enteric glia disruption is an inciting event in NEC
can be inhibited by the nucleotide-​binding oligomeriza- development might also explain in part why abdomi-
tion domain-​containing 2 (NOD2) receptor, previously nal distention and vomiting are seen so early in NEC,
known as caspase recruitment domain-​containing pro- given that these symptoms can be attributable to enteric
tein 15 (CARD15)64. NOD2 is an intracellular pattern nervous system dysfunction and intestinal dysmotility68.

Healthy premature infant Infant with NEC

TLR4 activation • Platelets

Bacteria 1 • Inflammasome
Amniotic fluid • Methylation
TLR4 Breast milk
• Prematurity
• Formula feeding C34
• Hypoxia NOD2
Lumen NEC

Constricted
1 Enterocyte vessels
apoptosis Mesenteric
vasoconstriction,
2 Impaired impaired perfusion
restitution 2
3 Reduced
proliferation Reduced eNOS
4 TH17 3
lymphocyte
Enterocytes induction Endothelial
4
5 Bacterial TLR4 activation
translocation

Fig. 1 | a model for the pathogenesis of neC based on bacterial signalling accumulation of pro-​inflammatory lymphocytes, which contribute to the
through tlr4 on the intestinal epithelium of the premature intestinal barrier injury through the release of IL-17. The subsequent barrier breakdown
epithelium. In the healthy intestine (left panel), bacterial colonization of the facilitates translocation of gut bacteria into the underlying vascular network,
gastrointestinal tract occurs without inducing an inflammatory response. By which can interact with TLR4 on the lining of the endothelium, leading to
contrast, in the premature gut (right panel), Toll-​like receptor 4 (TLR4) reduced endothelial nitric oxide synthase (eNOS), mesenteric
expression is elevated due to its role in regulating normal intestinal stem cell vasoconstriction and intestinal ischaemia. These processes are compounded
differentiation. Exaggerated TLR4 signalling in response to a dysbiotic by the effects of platelet dysfunction, DNA methylation and inflammasome
microbiome leads to intestinal epithelial cell death by apoptosis and activation, which have important roles in the development of necrotizing
necroptosis, as well as impaired mucosal restitution and reduced enterocolitis (NEC). TH17 , T helper 17. Adapted from T. Phelps, © Johns Hopkins
proliferation, leading to gut barrier injury. TLR4 signalling also induces the University, Department of Art as Applied to Medicine 2020.

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Although these findings reflect work from our group, interacting with the Wnt and Notch pathways in small
they have been confirmed by studies from other labora- intestinal stem cells11,12,63. TLR4 expression within the
tories in mice, rats, piglets and human tissue. Specifically, intestinal mucosa is very high in the premature gut com-
Caplan and colleagues first showed that C3H/Hej mice pared with the full-​term bowel11,35,75, reflecting in part
with an inactivating mutation in Tlr4 have reduced the developmental role of TLR4. We further determined
NEC incidence in a model of formula feeding and cold that the in utero developmental role of TLR4 switches to
asphyxia compared with TLR4 wild-​type C3HeB/Fej a pathological role in the setting of premature birth, as
mice10. Lu et al., also from the Caplan laboratory, showed activation of the persistently elevated TLR4 by bacteria
that the administration of polyunsaturated fatty acids within the infant intestinal lumen that are rich in TLR4
reduces the incidence of NEC in a neonatal rat model ligands leads to mucosal injury, intestinal ischaemia (see
and inhibits TLR4 gene expression69. Chan et al. showed below) and NEC. This concept of ‘TLR4 switching’ pro-
that TLR4 expression is elevated in rats with NEC and vides important mechanistic insights into NEC develop-
that an LPS-​neutralizing peptide that prevents TLR4 sig- ment and partly explains why NEC develops with greater
nalling attenuates NEC pathology in the rat jejunum70. frequency in the premature than in the full-​term host80.
Liu et al. showed that TLR4 expression in the intestine Although most evidence supports the link between
of newborn rat pups with NEC precedes evidence of his- TLR4 activation and NEC development in premature
tological injury, providing further support for the role infants, some studies have countered this hypothe-
of TLR4 in NEC development71. In a different neonatal sis. Szebeni et al. found that of 118 infants with a very
NEC rat model, TLR4 mRNA expression was found to low birthweight, 41 developed NEC, and there was no
be increased, with increased apoptotic intestinal epithe- association between TLR4 polymorphisms and disease
lial cells and markedly impaired proliferation72. More development81. White et al. developed a model of NEC
recently, Sun et al. confirmed that TLR4 expression is that involves Paneth cell disruption in mice, which
increased in neonatal mice with NEC and that sodium was found to occur independently of TLR4 signalling,
butyrate limits NEC severity whilst also reducing TLR4 as Tlr4−/− mice had similar NEC severity to wild-​type
expression73. In the preterm piglet NEC model, Yan et al. mice82. This finding was not unexpected, given that
showed that supplementary bovine colostrum feed- TLR4 expression was not increased in this particular
ings reduce NEC and reduce pro-​inflammatory gene NEC model and that NEC was induced at an older age
expression, including TLR4 in the intestine74. In human than in other studies (p14 versus p7), at which point lev-
studies, Shaw et al. performed shotgun metagenomic els of TLR4 in the intestine are quite low75. Moreover,
sequencing and quantitative PCR to characterize the fae- earlier studies in the piglet NEC model showed no
cal microbiota community of infants before NEC onset increase in TLR4 expression in the piglet intestine83,
and determined that infants who develop NEC show an although subsequent studies have revealed that TLR4
increase in microorganisms expressing the TLR4 ligand is increased over threefold in the presence of enteral
lipopolysaccharide (LPS; such as Enterobacteriaceae feeding84 and supplemental bovine colostrum feeds were
species)13, supporting the role of TLR4 in NEC patho- found to reduce both NEC and TLR4 expression in the
genesis. Interestingly, this study also identified another piglet intestine74.
group of infants with NEC who have low levels of CpG
DNA, which is the ligand for TLR9 and is an effective Clinical evidence for TLR4. Results from two studies in
TLR4 inhibitor75. Klerk et al. used human DNA extrac- human infants in the natural setting provide additional
tion from stool samples of premature infants and showed support for the role of TLR4 in NEC development. First,
that TLR4 methylation is increased in infants with NEC, a study in 550 infants with a very low birthweight who
providing additional evidence for the role of TLR4 in received human milk on over 50% of hospital days had
human disease76. In a thorough cataloguing of the tran- significantly lower rates of NEC (3.4% versus 13.5%) and
scriptional profile of human and murine NEC intestines, mortality (1.0% versus 4.2%) than infants who received
Cho et al. showed that TLR4 is increased at least twofold human milk on fewer than 50% of hospital days85. These
in both mice and humans compared with controls with- findings were confirmed by Hair et al. in a study in 1,587
out NEC, which they concluded is an important factor infants in four US centres: administration of an exclu-
in driving the pro-​inflammatory phenotype77. These sive human milk-​based diet substantially reduced NEC
findings support a role for TLR4 in NEC pathogenesis, incidence from 16.7% to 6.9%86. A systematic review
as has been reviewed by Gomart et al., who also linked and meta-​analysis that examined 49 studies showed that
TLR4 signalling with the TGFβ–Wnt/β-​catenin pathway human milk provides a clear protective benefit against
to explain its role in driving NEC development78. NEC, with an overall 4% reduction in incidence87.
Although these findings provide insights into the Interestingly, Good et al. found that breast milk inhibits
role of TLR4 in the pathogenesis of NEC, they also raise TLR4 signalling in the premature gut in mice and cul-
questions regarding how TLR4, a molecule that is typi- tured enterocytes as revealed by reduced TLR4-​induced
cally associated with protecting the host from invading NF-​κB via epidermal growth factor (EGF) activation of
pathogens79, could instead lead to disease development. the PI3K–AKT pathway88. Breast milk has been found
In addressing this apparent enigma, additional experi- to further reduce TLR4 signalling via its constituent oli-
ments have shown that TLR4 also serves an important gosaccharides, which Sodhi et al. showed can inhibit the
role in the normal development of the small intestine. LPS binding site on TLR4 and therefore dampen intes-
Specifically, Sodhi et al. from our group showed that tinal inflammation89. These studies were performed in
TLR4 signalling regulates normal gut development by cultured cells and confirmed in mouse models and with

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human NEC samples89. Furthermore, the intestine of is substantially different from the microbiome in pre-
the premature infant, in which TLR4 is expressed at very mature infants who do not develop NEC, and that these
high levels, is bathed in amniotic fluid that is swallowed differences include an increase in bacteria that are capa-
during fetal development90. In a surprising similarity ble of activating TLR4, such as Enterobacteriaceae13,99–101.
with breast milk, Good et al. found that amniotic fluid, It is noteworthy that the mode of delivery affects the
which is known to be enriched in EGF91, inhibits TLR4 infant microbiota, as the early microbiota of vaginally
on the intestinal epithelium via the EGF receptor92. These delivered infants resembles the mother’s vaginal micro-
findings were obtained in studies in which amniotic biota, whereas the microbiota of infants delivered by
fluid was micro-​injected into the fetal gastrointestinal caesarean section resembles microorganisms found on
tract of mice (on embryonic day 18.5) and confirmed in human skin102. One of the earliest studies that examined
intestinal tissue from mice and humans with NEC92. In the faecal microbiota of premature infants with and
additional proof-​of-​concept studies, Good et al. found without NEC was performed by Claud and colleagues103
that oral administration of amniotic fluid prevents NEC in a study of 20 premature infants (including ten with
development in mice and piglets92. Although the above NEC), who showed that infants with NEC had reduced
studies were from the Hackam laboratory, we note that bacterial diversity and an increase in abundance of
others have shown that infection of the amniotic cavity Gammaproteobacteria. In a subsequent study by Neu
with Gram-​negative bacteria (also known as chorioam- and colleagues, alterations in the microbiome of the
nionitis), which would be expected to lead to persistent intestine of premature infants occurred several days
TLR4 activation, is a significant risk factor for increased prior to the development of NEC and were character-
NEC severity in patients, with a threefold increase ized by an increase in abundance of Proteobacteria and
in NEC development in a meta-​analysis of 33 studies93. a decrease in abundance of Firmicutes, suggesting that
Taken together, these findings provide evidence to sup- these changes in bacteria may be causative as opposed
port a critical role for TLR4 in NEC pathogenesis, and to a consequence of NEC development104. A follow-​up
help illustrate the importance of breast milk and amniotic systematic review and meta-​analysis involving nearly
fluid in preventing NEC through effects on TLR4. 3,000 samples from 106 infants with NEC and 278 con-
trols confirmed these general findings, and concluded
Immature vascular system signalling. One of the strik- that the faecal microbiota from preterm infants with
ing clinical and pathological findings seen in patients NEC showed increased Proteobacteria and decreased
with NEC is the marked intestinal ischaemia that char- abundance of Firmicutes and Bacteroidetes before NEC
acterizes this disease. Several groups have investigated onset105. In addition to their role as activators of TLR4,
the pathways that lead to intestinal ischaemia and have the bacteria within the lumen of the gastrointestinal tract
uncovered important roles in NEC pathogenesis, lead- of premature infants have important roles in the mainte-
ing to potential opportunities to reverse the processes nance of gut homeostasis, through effects on metabolism
involved. For instance, Yazji et al. have shown that the and digestion, on the provision of energy to the intestinal
intestinal ischaemia in NEC occurs in response to mucosa in the form of short-​chain fatty acids, on intesti-
the translocation of bacteria across the damaged intes- nal motility and on other biochemical pathways within
tinal barrier, which activates TLR4 on the mesenteric the host106, all of which could explain the contribution of
endothelium, leading to a reduction in the expression bacteria to NEC development. In a metagenomic anal-
of the nitric oxide-​generating enzyme eNOS in mice67. ysis of 1,163 faecal samples from 34 premature infants
The TLR4-​mediated loss of eNOS expression tips the who developed NEC and 126 premature infants without
balance towards vasoconstriction of the mesenteric ves- NEC over a 5-​year period, a machine learning algorithm
sels, leading to the development of intestinal ischaemia67. revealed that samples that were collected before NEC
Additional studies have demonstrated that abnormal sig- revealed substantially more Klebsiella species, as well as
nalling through the vascular endovascular growth factor bacteria encoding fimbriae and bacteria encoding sec-
(VEGF) receptor 2 is important in NEC pathogenesis23 ondary metabolite gene clusters in the infants with NEC
and that VEGF plasmid injection decreases NEC severity as compared with the infants without NEC107. This study
in preclinical models94. Interestingly, polymorphisms in also showed that replication rates of all bacteria, espe-
VEGF are associated with increased NEC development cially Enterobacteriaceae that can activate TLR4, were
in humans95,96. The development of the vascular net- substantially higher 2 days before NEC diagnosis107.
work in the intestine depends on the gut micro­biome, as The link between an abnormal intestinal microbiota
germ-​free mice were shown to have an immature intes- and the development of NEC has led to an interest in
tinal capillary network compared with non-​germ-​free the study of probiotics in NEC prevention. As defined
animals97. These findings illustrate that the interplay by the Food and Agriculture Organization of the UN
between bacteria within the intestinal lumen and the and WHO, probiotics are “live microorganisms which
underlying immature vascular endothelial network can when administered in adequate amounts confer a
lead to impaired intestinal perfusion in the pathogenesis health benefit on the host”108. Indeed, a meta-​analysis
of NEC. published in 2014 of 20 randomized controlled trials
demonstrated that probiotics (focusing on Lactobacillus
The microbiome of the premature gut. The intestinal and Bifidobacterium species) reduced the incidence of
microbiota in patients with NEC plays an important severe NEC and all-​cause mortality109. A more recent
role in disease development98. Early studies revealed meta-​analysis that included 45 trials with 12,320 parti­
that the gut microbiome in premature infants with NEC cipants by Chi et al. showed that supplementation with

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Bifidobacterium plus Lactobacillus was associated with Treg cells in the intestine were reversed by administering
lower mortality rates (risk ratio 0.56) and NEC mor- the probiotic Lactobacillus reuteri in mice119, reveal-
bidity (risk ratio 0.47) than placebo supplementation110. ing the dynamic nature of the lymphocyte response in
However, there remains a degree of uncertainty regard- the pathogenesis of NEC.
ing the precise role of enteric bacteria in the pathogenesis Several studies have provided evidence supporting
of NEC and indeed whether changes in intestinal bac- a role for anti-​inflammatory, myeloid-​derived suppres-
teria are merely consequences of disease development. sor cells (MDSCs) in the pathogenesis of NEC. Levels
For instance, the characteristic intestinal bacteria seen in of MDSCs were increased in the cord blood and the
patients with NEC is highly variable, and there are also peripheral blood in a study of 71 preterm infants120,
reports that patient samples may have no statistically where they have been shown to exert anti-​inflammatory
significant differences in the pattern of microbial colo- and antibacterial roles121. Infants with a very low birth-
nization in premature infants with and without NEC111. weight (<1,500 g) who are prone to NEC had lower lev-
Moreover, the degree of similarity in the microbiota in els of MDSCs than infants with a normal birthweight
the infant’s intestine as compared with the various ani- (>2,500 g)121, whereas other myeloid cells could be
mal models is also highly variable112, adding additional converted into MDSCs and could protect mice from
complexity to this area of research. A final consideration NEC122,123. Notably, breast milk contains MDSCs, which
in evaluating the role of microbiota in the pathogenesis suggests that direct delivery could maintain tolerance in
of NEC is that most of the studies that have examined the gastrointestinal tract of nursing infants and further
a link between the microbiota of the premature gut and explain the protective role of breast milk against NEC124.
NEC development have relied on non-​culture-based Neutrophils themselves may have a causative role in NEC
bacterial sequencing techniques, which can often through the formation of neutrophil extracellular traps
lead to highly variable results113. More studies are needed (NETs), which serve to clear pathogens, and yet which
to clarify the role of the gut microbiota in NEC. may have a pro-​inflammatory role by priming other
immune cells to induce inflammation125,126. In an experi­
Other immunological players. The clinical manifes- mental NEC model, induction of polymorpho­nuclear
tations of NEC in infants stem from the development leukocytes worsened NEC severity127. These findings
of overwhelming local and systemic inflammation, broaden our understanding of NEC development by
suggesting that inflammatory cells (including lympho- revealing that bacterial–epithelial signalling leads to
cytes and neutrophils) might have key roles in disease both disruption of the intestinal epithelium and recruit-
pathogenesis114. In assessing the role of lymphocytes in ment of pro-​inflammatory cells that together cause the
NEC pathogenesis, Weitkamp et al. found that in human inflammatory response seen in infants with NEC.
NEC tissue, the ratio of anti-​inflammatory regulatory T
(Treg) cells was significantly lower in patients with NEC Other pathways. In addition to studies on bacterial sig-
than in age-​matched controls115. To further investigate nalling in the pathogenesis of NEC already described,
the potential role of inflammatory cells in the pathogen- several additional pathways have been implicated in
esis of NEC, Egan et al. from our group examined the NEC pathogenesis in mice and humans. Several groups
cellular constituents of the small intestines of newborn have provided evidence that impairments in the coagula-
mice and premature humans with and without NEC, and tion cascade and the haematological system play critical
found that mouse and human newborn intestinal epithe- parts in NEC development. Specifically, the activation of
lium in NEC is characterized by a significant accumu- neonatal platelets by thrombin in response to tissue fac-
lation of lymphocytes35, which on further analysis were tors released from intestinal macrophages causes intesti-
found to be TH17-​expressing RORγt cells35. The adop- nal injury that can be reversed by thrombin inhibition in
tive transfer of lymphocytes from the intestines of mice mice128. The clinical relevance of this finding is shown by
with NEC into naive mice resulted in the spontaneous the finding that circulating tissue factor and thrombin–
induction of NEC in recipient mice, indicating that these antithrombin complexes are increased in patients with
pro-​inflammatory T cells have a causative rather than a NEC128. Earlier studies by the same group revealed that
secondary role in NEC development. Importantly, intes- the administration of erythrocytes to anaemic mice has
tinal epithelial TLR4 signalling resulted in the upregu­ a role in NEC development through a mechanism that
lation of CCL25 on the intestinal epithelium, leading involves TLR4 activation129. The intestinal inflammation
to the recruitment of naive lymphocytes, which differ- seen after erythrocyte transfusion depends on TLR4
entiated into TH17 cells after the secretion of IL-6 and signalling in macrophages, as macrophage depletion
IL-1 by the inflamed epithelium, whereas numbers of also attenuates the transfusion effect on the intestine129.
anti-​inflammatory Treg cells were reduced35. The subse- These experimental findings could explain haematolog-
quent release of IL-17 from the FOXP3+ lymphocytes ical predictors of NEC in patients, including erythrocyte
induced the loss of tight junctions between adjacent transfusion and platelet dysfunction3, and are supported
enterocytes, promoting bacterial translocation35, while by additional models indicating that platelet dysfunction
antibodies to IL-17 and IL-17R inhibition prevented the can lead to NEC-​like lesions in mice130,131.
intestinal injury in NEC35. Additional investigators have A variety of studies have pointed to the role of DNA
confirmed these findings77,116,117, and additional stud- methylation in NEC pathogenesis. A methylome signa-
ies have shown that neonates with NEC before day 14 ture associated with NEC was detected in the stool of
exhibit higher expression of CCR9 in CD4+ T cells on infants with NEC, providing clues to NEC pathogenesis
day 14 (ref.118). Interestingly, the reduction in FOXP3+ through global changes in gene expression that might

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be tied to disease development132. Additional studies (CCL25) expression within the lining of the airways,
have shown that DNA methylation of TLR4, VEGFA which results in the recruitment of pro-​inflammatory
and DEFA5 is associated with NEC in preterm infants, TH17 lymphocytes to the lung, which exacerbates the
consistent with the findings regarding the role of TLR4 NEC-​induced lung injury16. Moreover, the inflam-
and VEGF in disease pathogenesis76. Moreover, stud- matory response in the lung results in a reduction in
ies in mouse models133 and surgically resected human eNOS, which would reduce perfusion, making the lung
tissue133–135 have shown an important role for inflammas- injury worse137. Other mechanisms that might explain
ome activation and NF-​κB signalling in NEC, findings the development of NEC-​associated lung injury include
that could explain the broad systemic inflammatory a loss of endothelial nitric oxide synthase138 as well as
response often seen in patients with this disease133–135. activation of pro-​inflammatory lymphocytes and macro­
These findings reveal that NEC is a complex disorder phages that become activated in the inflamed gut, and
arising from initial bacterial signalling events, result- that can secondarily injure the lung139. These mecha-
ing in tissue injury through effects of haematological nistic insights were derived from studies in mice, and
impairment, methylation effects and inflammasome additional studies using human tissues or pulmonary
signalling. leukocytes will need to be performed for clinical valida-
tion. Minimal direct clinical evidence supporting these
Influence of NEC on the lung and brain. Children who mechanisms in terms of lung injuries exists, partly due to
survive NEC often have long-​term complications, the the difficulty in obtaining human lung samples for study
most clinically significant of which affect the lungs and in this clinical situation.
the brain. Lung disease in children with NEC is more Patients with NEC can also develop severe neuro­
severe than the lung disease in premature infants who logical dysfunction that occurs in 24% to 55% of infants
do not have NEC, and is characterized by persistent and with NEC140,141, and which affects their learning, memory
prolonged ventilator requirements7,136 (Fig. 2). Jia et al. and social interactions8,39,40,142. NEC-​associated brain
have shown that lung injury in NEC results in part from injury is notably more severe than the brain injury seen in
the activation of TLR4 on the pulmonary epithelium, as premature patients who do not develop NEC8,39,40,142.
deletion of TLR4 from the pulmonary epithelium pro- In a mouse model that mimics the brain injury seen in
tected the lungs in mice with NEC18. Mechanistically, human infants, Nino et al. found that NEC-​associated
the activation of TLR4 on the lining of the premature brain injury requires the TLR4-​dependent release of
gut induces the release of high-​mobility group box 1 HMGB1 from the inflamed intestine17. HMGB1 release
(HMGB1) from the newborn intestine, which activates from the intestinal epithelium activates TLR4 on the
pulmonary epithelial TLR4, leading to the induction of microglia, leading to the release of reactive oxygen spe-
the neutrophil-​recruiting C-​X-​C- motif chemokine 5 cies, a reduction in oligodendrocyte progenitor cells,
(CXCL5) and the influx of pro-​inflammatory neutrophils and the subsequent loss of myelin in mice17. Importantly,
to the lung in mice18. TLR4 activation on the pulmo- the oral administration of a ROS scavenger fused with
nary epithelium also induces C-​C motif chemokine 25 a dendrimer designed to target the microglia prevents
myelin loss and reverses neurological dysfunction17.
Zhou et al. described how IFNγ-​producing lympho-
HMGB1 Epithelial cytes traffic from the inflamed intestines to the brain
CXCL5
release TLR4
in mice15, which compounds the brain injury. These
Neutrophils findings define a previously unexplored mechanism
whereby NEC leads to lung and brain injury through
Inflamed Lung injury shared cellular processes involving the release of fac-
TLR4 intestine
in NEC tors from the gut (such as HMGB1), which can activate
TLR4 on the secondary organs (such as the pulmonary
epithelium and the microglia), and to the release of
pro-​inflammatory lymphocytes. Additional mechanisms
proposed to explain the development of NEC-​induced
Gut lymphocytes IFNγ
brain injury include disruption of normal brain devel-
opment as a result of impaired perfusion, metabolic defi-
Microglia OPC Myelin ciency, hypoxic damage143 and exaggerated activation
HMGB1 ROS Brain injury
activation loss loss of the NLPR3 inflammasome133. These findings might
provide insights not solely towards our understanding
Fig. 2 | necrotizing enterocolitis beyond the gut. Infants with necrotizing enterocolitis of the development of NEC but also for other diseases
(NEC) develop long-​term morbidities that affect the lung (red) and the brain (grey). in which the innate and adaptive immune systems are
Mechanistically, Toll-​like receptor 4 (TLR4) activation in the inflamed intestine leads to linked in the development of organ dysfunction in the
the release of high mobility group box 1 (HMGB1) from the intestinal epithelium, which
newborn baby.
activates TLR4 on the pulmonary epithelium, leading to the recruitment of the neutrophil-
recruiting C-​X-​C motif chemokine 5 (CXCL5) and the influx of pro-​inflammatory
neutrophils to the lung causing injury. In parallel, HMGB1 release from the gut leads to Potential maternal–fetal origin. The fact that pre­
activation of TLR4 on the microglia of the brain, leading to release of reactive oxygen maturity is central to the pathogenesis of NEC raises
species (ROS) that cause a loss of oligodendrocyte progenitor cells (OPCs), and myelin the possibility that factors present in the maternal
loss that causes brain injury. In parallel, lymphocytes traffic from the gut to the brain and environment might have a potential role in subsequent
release IFNγ, causing further brain injury. NEC development. In support of this notion, Lu et al.

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showed that during pregnancy, the administration of a understanding of the pathways that lead to NEC, var-
diet rich in the aryl hydrocarbon receptor (AHR) ligand ious new potential treatment strategies for NEC have
indole-3-​carbinol, which is present in green leafy vegeta- been identified in the past several years. These potential
bles, prevents NEC in newborn mice by reducing TLR4 therapies can be broadly divided into probiotics, small
signalling in the newborn gut of mice14. These findings molecules and biologic agents.
were confirmed in human NEC tissue and shed addi- The efficacy of probiotics for NEC prevention remains
tional light on the predisposition of the premature new- an exciting area of research, and several large trials
born to NEC, as AHR levels are low and TLR4 is high have shown their protective role in NEC150. As already
in the premature intestinal epithelium14. In support of mentioned, a meta-​analysis published in 2021 showed
a potential clinical role for these findings, breast milk that supplementation with Bifidobacterium plus Lacto­
was found to be rich in AHR ligands, providing fur- bacillus was associated with lower rates of mortality (risk
ther insights into how breast milk could inhibit TLR4 ratio 0.56) and NEC morbidity (risk ratio 0.47) than pla-
to prevent NEC14. The mechanism of TLR4 inhibition cebo supplementation110. The mechanisms of action by
by AHR activation in the diet in mice involves the which probiotics prevent NEC remain debated, but is
upregulation of microRNAs that reduce TLR4 expres- thought to involve a restoration of the luminal bacteria
sion in the gut, namely miR-146b, miR-223 and let-7i towards more anti-​inflammatory species151. Additional
in wild-​type mice14. In terms of the physiological rel- studies have revealed that the protective role of probio­
evance of these findings, previous investigators found tics might also involve the activation of TLR9 by bac-
that the administration of a Mediterranean diet, which terial DNA, which inhibits TLR4 signalling within the
is rich in AHR ligands, to 82 pregnant mothers pre- intestine via IRAK-​M upregulation, an effect that was
vented prematurity and NEC144,145. Other intrauterine observed in mice and piglets75,152.
factors that have been implicated in the development of There has been a range of small molecules that have
NEC include prenatal medication exposure, including been shown in preclinical studies to prevent or treat
amoxicillin-​clavulanic acid, which was associated with NEC. Our group identified a novel TLR4 inhibitor,
an odds ratio of 2.3 for NEC development146, and indo- namely C34, as a member of a novel class of oligosac-
methacin which was associated with an odds ratio of 7.1 charides that prevent and treat NEC in mice and piglets
for NEC development147, whilst antenatal steroids reduce when administered orally or intravenously, therefore
the risk of NEC (risk ratio 0.5)148 through processes that opening the door to potential clinical use153. Additional
remain ill-​defined149. Furthermore, in a meta-​analysis translational development for this TLR4 inhibitor
including a total of 22,601 pregnant women, a clinical includes studies showing that C34 can block TLR4
diagnosis of intra­uterine infection increased the risk and thus prevent acute lung injury after trauma154, and
of NEC with an odds ratio of 1.24, and in a subgroup restore levels of anti-​inflammatory lymphocytes in the
analysis, the presence of umbilical cord inflammation lung and thus attenuate NEC-​associated lung injury16. In
was associated with a greater than threefold increase parallel, we found that the administration of the NOD2
in the risk of NEC93. By broadening the focus of NEC agonist MDP upregulates the molecule SMAC-​diablo,
research towards earlier time points, including the pre- and therefore prevents NEC through secondary inhibi-
natal period, we might gain new insights into the mech- tion of TLR4 (ref.64). The potential clinical role of NOD2
anisms of this disease and identify novel treatment agonists for NEC prevention is supported by data show-
approaches. ing that NOD2 loss-​of-​function mutations increased
the risk of NEC in a European cohort of 9,429 infants
Preclinical therapies with a very low birthweight (<1,500 g)155. The discovery
At present, there is no specific treatment for NEC, and of the maternal–fetal inflammatory axis in the develop-
current therapies — including antibiotics, cessation of ment of NEC through activation of AHR has led to the
oral feeds and surgical resection of necrotic bowel — identification of AHR ligands that can be administered
are often provided after the window for reversibility has to the mother during pregnancy in preclinical models,
closed (Box 1). Furthermore, although the administration resulting in reduced TLR4 signalling and decreased NEC
of breast milk has tremendous benefits in preventing in the offspring14. Our identification of a critical role for
NEC, it is not available for all infants, especially after the impaired enteric nervous system in NEC pathogen-
premature birth. However, based upon an improved esis in mice and piglets, and confirmed in human NEC
tissue, has led to the identification of a small molecule
that can activate enteric glia to release BDNF, which lim-
Box 1 | Current therapeutic approaches for neC
its NEC severity in mice and reducing inflammation in
• Early-​stage necrotizing enterocolitis (NEC): fluid and human intestinal resection specimens68. In addition, the
inotrope resuscitation; broad-​spectrum antibiotics; finding that intestinal ischaemia in NEC results from
cessation of feeds. decreased eNOS activity in the mesenteric endothelium
• Later stages of NEC: surgical resection of necrotic has led to successful preclinical studies showing that
intestine, drainage of the abdominal cavity. nitric oxide donors and precursors can prevent NEC
• Preclinical therapies in development: Toll-​like receptor through enhanced perfusion67. A large body of work has
4 (TLR4) inhibitors; aryl hydrocarbon receptor agonists; shown that HBEGF prevents NEC by reducing inflam-
stem cell therapies (intestinal and mesenchymal); mation and enhancing mucosal healing156, while the use
synthetic amniotic fluid; breast milk exosomes; of exosomes from intestinal stem cells offer additional
anti-​cytokine therapy; growth factor therapy.
approaches for NEC prevention and treatment157,158,

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Box 2 | Future outlook and outstanding questions for NEC prevention in patients that is safe, in part
because of the high amounts of IgA that are normally
• What are the Toll-​like receptor 4 (TLR4)-​dependent pathways that lead to necrotizing present in breast milk162. Other studies have provided
enterocolitis (NEC)? insights into the potential role that stem cells might offer
-- Define the factors that regulate TLR4 signalling, and identify in a cell-specific manner in the treatment of NEC163–165 by enhancing the regener-
TLR4-​dependent pathways that induce NEC.
ation of the intestinal epithelium, as well as tantalizing
• What are the effects of gut metabolism on NEC induction?
-- Define how impaired metabolism in the premature intestine leads to NEC, and how evidence that remote ischaemic preconditioning — a
modification of metabolic pathways prevents NEC. technique by which ischaemia is briefly induced in
• Can we predict NEC earlier so as to prevent disease? a limb — can attenuate NEC in mice through nitric
-- Development and integration of advanced intestinal imaging techniques, novel oxide release and vasodilation in the gut166.
biomarkers and genetic risks to predict NEC. How do we integrate these potential therapeutic
• Can we prevent the long-​term complications of NEC on the lung, the brain and the gut? approaches into a unifying NEC prevention or treatment
-- Understanding how inflammation affects the developing brain, lung and intestine, model? Given the heterogeneity that exists in patients
to protect these secondary organs. who present with NEC, it is possible that some thera-
peutic approaches will be more suited to certain patients
through the delivery of cargo that includes mRNAs that over others (such as a TLR4 antagonist in a patient with
can enhance mucosal healing159,160. very extensive disease as compared with an IL-17 inhib-
The discovery and application of biologic therapies itor in a patient with early disease), or that certain agents
for NEC remain an area of interest. These include the might be more useful for prophylaxis (such as an AHR
use of anti-​IL-17 and all-​trans-​retinoic acid, which serve agonist, or maternal IgA) rather than for treatment. We
to restore the balance between the pro-​inflammatory posit that the prevention and treatment of NEC will not
TH17 and the anti-​inflammatory Treg cell environment35, be achieved using a single agent but rather through a
a finding supported by an earlier observation showing multipronged approach, using combinations of these
that the administration of anti-​TNF agents can reduce potential therapies. The fact that there are many poten-
NEC in mice161. Maternal IgA can protect against NEC in tial treatments on the horizon provides a reason for cer-
mice through binding to faecal bacteria, suggesting that tain optimism. Translation of these preclinical findings
administration of IgA might offer a biological approach to the clinic will first require regulatory approval, which
can be difficult when premature infants are involved
due to concerns for infant safety167. Subsequently, multi­
1 Search for TLR4-dependent 2 Discover effects of metabolism centre randomized trials will be required, with a focus
molecular pathways on the prevention of NEC on end points that include reduction in NEC devel-
opment and progression of mild to severe NEC. That
only approximately 10% of premature infants develop
NEC raises two considerations for clinical trial devel-
opment. First, a large number of patients will need to be
included to develop a study of sufficient power to detect
differences between groups and is likely to require multi-
institutional collaborations to be successful. The NEC
Healthy premature Premature infant trial by Blakely et al. published in 2021 is an example
AHR ligands
infant with NEC of one such successful collaboration, in which random-
ization of a surgical approach for the treatment of NEC
3 Predict NEC development 4 Prevent long-term was tested in a consortium of multiple neonatal inten-
complications of NEC sive care units168. The second consideration pertains to
Spectral Gut-derived the fact that since the overall incidence of NEC is low
imaging molecules compared with the number of premature infants who do
not develop NEC, any potential therapeutic intervention
Stem must have extremely low toxicity, especially in such vul-
Maternal cells nerable patients. A greater understanding of the patho-
factors
physiological pathways that lead to NEC can potentially
Lymphocyte
NEC address these potential barriers by tailoring an individual
treatment to a specific pathway in a particular patient169.
Machine learning
• Microbial taxa Artificial Future outlook
• Microbial metabolism intestine Whilst a great deal of ground has been gained in our
understanding of the underlying pathophysiology of
Fig. 3 | Key areas of research focus in the field of neC. The field of necrotizing NEC, we still lack a specific treatment and are still largely
enterocolitis (NEC) has seen major advances over the past decade. To move the field
unable to determine which babies will develop NEC and
forward, four major areas of research need to be addressed. These include (1) a search for
Toll-​like receptor 4 (TLR4)-​dependent molecular pathways, (2) understanding the effects
which will not. This sobering reality leads us to outline
of metabolism on NEC prevention, (3) understanding how to predict NEC including a key focus areas for further study in the field (Box 2;
focus on maternal factors in NEC development, and (4) understanding the long-​term Fig. 3). First, based upon the important parts played by
complications of NEC. AHR, aryl hydrocarbon receptor. Adapted from T. Phelps, TLR4 signalling in the pathogenesis of NEC, we will
© Johns Hopkins University, Department of Art as Applied to Medicine 2020. need to conduct a detailed search for TLR4-​dependent

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molecular pathways that could lead to NEC develop- Conclusions

ment. These studies will need to be accompanied by NEC is a complex disease that continues to cause death
a detailed analysis of functional bacterial genomics, and disability in many premature infants. In seeking to
including their gene products, metabolites and poten- understand the molecular underpinnings of NEC, we
tial interactions with TLR4 and other bacterial recep- have advanced a unifying theory of NEC development
tors on host cells within the gut and in other tissues to that involves bacterial signalling via TLR4 in the prema-
define the critical pathways that lead to NEC develop- ture gut, which is substantially elevated as a consequence
ment. Other pathways beyond TLR4 will be important of its regulation of normal gut development in utero.
to consider, given the complex nature of NEC, including Additional studies have shed light on the role of NEC in
those that regulate pro-​inflammatory cells, metabolism inducing damage to the brain and lung, and have indi-
and the microbiome. Second, we need a broad approach cated an emerging role for the maternal environment in
to discover the effects of metabolism on the prevention NEC development. Findings from a variety of investiga-
of NEC, given in part the finding that AHR activation tors have led to the discovery of several classes of mole­
by ligands in food can reduce NEC severity and the cules that can prevent NEC through interfering with
nearly universal observation that NEC arises after host–microorganism signalling, in preclinical models.
the initiation of formula feeding. Third, there needs to Future studies in the field might focus on identifying the
be a focus on predicting which babies will develop NEC, effects of metabolism on NEC, improving NEC predic-
through advanced imaging techniques, an assessment of tion using artificial intelligence-​based approaches, fur-
maternal factors or the incorporation of machine learn- ther discovery of TLR4-​dependent signalling pathways
ing, so that early treatment can be initiated. Finally, we and preventing the long-​term complications of NEC
need to focus on the long-​term complications of NEC, development. Such studies are likely to shed light not
including the effects on the brain and the lung, and the just on the pathogenesis of NEC but also on the factors
development of short-​bowel syndrome, which arises that determine gut and immune development in general.
when substantial portions of the intestine are damaged As a result of these efforts, it is hoped that studies in this
and removed. Through a focus on these future areas of field will advance our ability to care not just for patients
study, we will learn not only about the factors that lead with NEC but also for those infants at risk of additional
to NEC, but will gain greater insights into the develop- diseases of prematurity that contribute to the morbidity
mental regulation of the neonatal immune system and seen in this vulnerable population.
the gut, and its interaction with the complex metabolic
and bacterial world in which it must develop and thrive. Published online xx xx xxxx

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