Necrotizing Enterocolitis (NEC) and Darwinism (Reviewing the Evolutionary Basis for NEC)
Phillip V. Gordon MD PhD
Department of Pediatrics, Tulane School of Medicine, New Orleans, LA
Phillip V. Gordon, MD, PhD
Department of Pediatrics
Tulane School of Medicine
1430 Tulane Ave
SL-37, Rm 5210
New Orleans, LA
Author Disclosure: The author has nothing to disclose.
There is a tendency for neonatologists to think of necrotizing enterocolitis (NEC) as a human specific disease, but all mammals are at increased risk for intestinal enterocolitis in the neonatal period. This manuscript reviews the passive immunity transfer mechanisms by which mammalian neonates survive the transition from innate to adaptive immunity. Context is provided for forms of neonatal mortality across common veterinary practices and the fundamental role of neonatal enterocolitis as an evolutionary screening mechanism is explored. Finally, potential areas of relevance and discord between the animal and human conditions will be considered as it relates to the practice of neonatology (in italics).
Key Words: necrotizing enterocolitis, mammal, evolution
Placentation is thought to have evolved roughly 90 million years ago, based on the fossil record and more recent mitochondrial DNA studies.1,2 A rudimentary characteristic of the early placenta was the capacity to synthesize pituitary hormones essential for fetal survival and growth. Comparative studies of mammalian and marsupial genomes are consistent with an evolutionary model known as the long fuse, in which the placental programming of progenitor mammals persisted largely unchanged until relatively recently in paleontological terms, at which time explosive diversification occurred.3-6 Today, one of the most dramatic examples of placental and mammary gland divergence is found in mechanisms of passive immunity transfer from the mother to the offspring (see Figure1).
Passive transfer of immunoglobulins
Evolution has derived three fundamentally different methods of transferring passive immunity from the mammalian mother to the fetus / neonate. In each case, the transfer
of immunoglobulin provides passive host defense, in combination with the innate immune system, until active adaptive immunity can be optimized by the neonate.
Passive transfer of immunoglobulins in colostrum
In many livestock (foals, calves, lambs), colostrum contains high concentrations of immunoglobulin and the neonatal intestine is transiently primed to uptake these antibodies using the FcRn receptor.7-10 Horse veterinarians maintain supplies of frozen mare colostrum for those cases where colostrum production fails, because the consequences of failure of passive immunity transfer (FTP) are severe.10 A recent study showed that foals with untreated FTP have high mortality, dying most commonly from either respiratory illness or neonatal enterocolitis because of subsequent hypogammaglobinemia.10 Of interest is the fact that after let down, the mare switches predominately to the secretion of IgA antibody, which is poorly absorbed, but thought to be critically important in passive host defense against enteric pathogens. Animals employing this narrow window mechanism of passive immunity transfer generally have only one or two offspring and thus have a high likelihood of all offspring getting sufficient amounts of colostrum, prior to milk let down.
Passive transfer of immunoglobulins in breast milk
Rodents have been best studied for uptake of immunoglobulins in breast milk. Their density of FcRn is highest in the proximal small intestine (then declines in a proximal to distal fashion towards the colon). During cell death, the FcRn on the dying cell surface has much higher immunoglobulin affinity.12 This arrangement may create a super-opsin situation as dying cells undergo anoikis and shed into the lumen, possibly augmenting the value of immunoglobulins that have attached to a pathogen. Unfortunately, this same property makes it very hard to do immunohistochemistry research with rodent models of NEC and calls into question some of the literature involving such models.
Because rodents have a short gestation / large litter reproduction plan, a more chronic period of immunoglobulin uptake may provide the greatest benefit to the greatest number of pups. FcRn mediated transfer is an active process, requiring ATP-mediated endocytosis with intracellular trafficking from the apical side, through the endoplasmic reticulum sorting apparatus and out the basal side of the enterocyte. It is unknown precisely how long this capacity lasts in rodents, but many aspects of rodent intestinal maturation are triggered by weaning.
Passive transfer of immunoglobulins across the placenta
Primates provide immunoglobulin transfer to the fetus via the placenta.13-15 This generally occurs late in gestation and contributes to the lower extremity swelling often observed during late human pregnancy (due to decreased oncotic pressure). Premature neonates are at increased risk of enterocolitis in no small part because they miss this passive infusion of immunoglobulin and then are subsequently infected by the very organisms that their mother already has an immune response to. One very impressive example of this phenomenon is the herpes virus.16 Term infants rarely are infected in mothers with a past history of herpes. In contrast, herpes is often fatal in late second trimester infants who are born to mothers who have reactivation at the time of delivery. Studies of empiric administration of pooled immunoglobulin to premature infants have been done, based on our understanding of passive immunity, but this therapy does not appear to be efficacious in preventing or rescuing infants from the morbidity or mortality of necrotizing enterocolitis (NEC).17-19 The reasons for this likely have to do with the novel nature of NEC as a disease that originates at the mucosal surface (a site with poor humeral immune surveillance). In contrast, immunoglobulins have been used to rescue livestock with FTP from respiratory illness.
Passive antibody transfer has evolved in three separate forms to help mammalian neonates survive the transition from innate immunity to adaptive immunity. Humans are dependent upon placental transfer, whereas nearly all animal models of NEC utilize intestinal transfer. This represents a major pitfall of basic NEC research, since intravenous administration of immuno-globulin does not appear to be helpful in human neonates. In contrast, IgA may be a very important component of NEC prevention at mucosal surfaces (and is depleted in pooled immunoglobulin).
Comparing enterocolitis in animal groups with humans
Human NEC versus neonatal enterocolitis in livestock:
Human NEC has been recognized for more than 50 years, but over time we have gradually redefined it until it has become specifically associated with the presence of bowel wall pneumatosis on x-ray and the attendant hemorrhagic necrosis on pathology (reviewed in reference 20). Animal studies and veterinary medicine have also reported a number of neonatal enterocolitis conditions in term animals.6-10 Foals are known to have a high fatality rate with Clostridia associated enterocolits (>50%).10 Piglets, lambs and calves have also been shown to have such an entity. Early Clostridia colonization has been associated with human NEC in both retrospective and prospective cohorts but is not associated with most cases in most studies.21 The magnitude of this last observation may be a function of how investigators look for Clostridia, as a recent piglet enterocolitis study suggests that the organism adheres to the intestinal wall (and thus can be detected by PCR amplification of pathology specimens) but often is undetectable in stool of animals and is only detected in humans at an uncommon frequency.22,23 In foals, Koch’s postulate was fulfilled when 90% of foals who ingested Clostridia spores acquired neonatal enterocolitis.11 Experimentally, Clostridia is the only species of bacteria ever shown to be able to generate pneumatosis (to the author’s knowledge).
Neonatal enterocolitis in term livestock is commonly associated with early Clostrida colonization and likely represents an important environmental stressor for determining survival of the fittest. A human NEC subset clearly correlates with this disease entity, but it remains unclear what the contribution of Clostridia is as a causative agent within the spectrum of human NEC as a whole.
Human NEC versus experimental models of NEC in animals:
Animal research into NEC has yielded a myriad of models in which a NEC-like enterocolitis (NECLE) can be induced. Rats, mice, and piglets are the most commonly used animals, although monkeys and lambs have also been used in limited studies. Common variables used to induce NECLE include feeding of formula instead of breast milk, hypoxia, cold-stress, aberrant bacterial colonization, and prematurity. While necrosis of the distal small bowel wall is universal, pneumatosis is variable and sometime even absent in these models (perhaps because they often do not specifically employ Clostridia colonization as a risk component). Many of these variables recapitulate natural stressors. In particular cold, hypoxia and aberrant colonization correlate well with cool ambient temperature, partial birth asphyxia and mastitis / aberrant enteric colonization in the wild. Prematurity in humans is the most important risk factor for NEC and has the highest degree of correlation with risk and severity.
The natural stressors of cold, partial birth asphyxiation, mastitis and prematurity may lead to NECLE in animals and correlate well with known risks for NEC in humans. Rapid death from NECLE likely preserves the life of siblings in larger litters by preventing prolonged maternal distraction, as well as that of the mothers themselves, who may be at risk for predation while tending a non-thriving neonate. In human neonates, preventing cold and hypoxic stress, promoting mother’s milk to optimize appropriate colonization, and understanding the role of prematurity are essential components of NEC prevention.
Fetal dependence upon innate immunity
In understanding that there is a requirement for passive immunity to support the transition from innate to adaptive immunity, it becomes clear that the mammalian fetus relies upon innate immunity for host defense in the first two thirds of gestation. The mother’s adaptive immunity continues to protect the fetus while she surrounds it in the womb, but this issue of immune-incompetency becomes paramount with extreme preterm birth. Such individuals are born without passive transfer of immunoglobulin and without adaptive immunity. Animals born at this gestational age die from surfactant deficiency, but humans in the modern world are rescued with artificial and animal derived surfactants.
To fully understand the implications of this new survival paradigm, one must understand how the innate immune system works. Toll-like receptors (TLRs) on the surface of intestinal epithelia recognize specific bits and pieces of pathogens. Upon activation, they set off a cascade of signals (nearly all of them activate the same pathway known as NF-kappa-B), resulting in programmed cell death. As the cell dies, it engulfs the pathogen, killing it at the same time. This works fine if it only happens occasionally, but what evolution and modern medicine have inadvertently done is stack the deck against premature neonates. TLRs increase in number with gestational age as a means of optimizing pathogen surveillance in utero (where the infant can heal any tissue damage caused by excessive apoptosis). Unfortunately, this doesn’t work so well ex utero.
The risk of NEC increases exponentially in proportion to the TLR density in preterm infants. The closer such an infant gets to the corrected gestational age of term, the greater the apoptosis response to an enteric pathogen and thus the greater the risk of NEC. Shortly before term, the adaptive immune system is primed, the TLRs are actively removed from the cell surface, their expression is halted, then the risk for NEC decreases dramatically. This quirk has created one big headache for neonatology over the last 25 years, because the timing of NEC seemed increasingly unpredictable after the introduction of surfactant. However, we may finally be able to understand NEC timing, because TLR biology seems to finally be correlating with NEC data (see Figure 2 for schematic illustrating this concept), now that we have removed the confounder of spontaneous intestinal perforations from our datasets (reviewed in reference 20).
Premature mammals typically die of surfactant deficiency but humans in the modern world rarely do, thus a new preterm NEC has emerged in the post-surfactant era. This entity is subtly different from the neonatal enterocolitis (and term NEC). It occurs at a different time in life and in the absence of both passive and adaptive immunity. Models like the one in Figure 1 may be useful in NEC prevention if they accurately predict the window of onset in preterm patients.
NEC / neonatal enterocolitis in the face of adaptive immunity
Most if not all veterinarian reports of neonatal enterocolitis in livestock involve term animals. Most models of NEC have been performed in term rodents or piglets. There have been a few heroic attempts in pigs and rodents that can be objectively categorized as late preterm when compared to humans, but these are rare and the issue has always been one of sustaining survival long enough to acquire NEC. Certainly neonatal enterocolitis, when it occurs naturally, is a disease of the term infant. Thus, this disease occurs in animals with adaptive immunity (although they may suffer from FTP). In humans, NEC in the term infant is associated with distinct risk factors including hypoxia at birth, over-feeding, formula feeding, maternal drug use, and congenital heart disease. These variables point to a hypoxic-ischemic impact upon the bowel (rather than an infectious trigger or immune system dysfunction). It is an extremely rare disease in comparison to preterm NEC and the enterocolitis found in livestock because human immunoglobulin transfer occurs prior to term birth. With regard to Darwinism, neonatal enterocolitis ensures the survival of the fittest by preserving the lives of siblings and the mother.
Neonatal enterocolitis and human term NEC are likely the same pathogenic entity, one that has surely been with mammals for our 90 million years of existence (since there are no known mammals who do not display the ability to acquire it). Its triggering requires external stressors (e.g. birth hypoxia or early Clostrida colonization) and/or immune dysfunction (FTP) to compromise the defense of the gut. Neonatal enterocolitis is relatively rare in incidence but probably serves an important evolutionary function in natural selection. Term NEC is even rarer as a clade specific event due to placental transfer of immunoglobulin and could be eradicated because its risk factors are known and can be avoided (i.e. formula feeding and over feeding).
Examining the human disease of NEC within the context of passive immune transfer evolution has intrinsic value. First, it demonstrates which subset of the human disease spectrum (term NEC) correlates best to the natural disease state (neonatal enterocolitis). It also reveals that, like veterinary medicine, we have it in our power to dramatically reduce the incidence of this specific disease subset. Second, it reveals a common theme of modern neonatology, namely we have created our own new version of enterocolits through the use of surfactant (preterm NEC). Third, in understanding how different mammalian clades evolved different passive immunity transfer solutions; one begins to understand why nature might find it advantageous to position an executioner at this developmental toll-bridge. Doing so increases the chance of sibling and maternal survival by minimizing wasted resources on non-viable progeny. However, because the mortality for NEC is (by evolutionary design) extremely high, the primary goal of current research should be prevention rather than therapy. Nature has predetermined that this will be the more successful approach.
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