Overview

The primary role of the gastrointestinal (GI) tract is to facilitate digestion and absorption of nutrients, but the intestines also engage in a multitude of other biologic functions, including significant immunologic and neuroendocrine activities. The intestinal surface is composed of a layer of epithelial cells that comprise villi, which provide a sizeable surface area to facilitate exposures to a vast array of food antigens, bacteria flora, and microbial metabolites.

The neonatal intestine functions during a period of major transition, including alterations in luminal and surface enzyme activities, changes in both the innate and adaptive immune systems, and major shifts in the microbiome, depending on environmental factors, such as diet and other exposures. In addition, premature infants may be still completing basic morphogenesis and development in addition to coping with the changes in their environments. There is new recognition that the neonatal intestine is exposed to microbes in utero and that this likely plays a major role in intestinal development, even before birth and subsequent exposure to the extrauterine milieu. This chapter will provide an overview of the intestinal mechanism that relates to systemic inflammation, the relationship and interactions between the intestinal microbiota and the intestinal mucosa, as well as the resultant effects on the developing nervous system.

Components and Functions of the Gastrointestinal Epithelium

The GI surface is composed of several types of cells. The epithelial layer originates from the endoderm and throughout gestation changes and differentiates through recanalization of the intestinal lumen and, later, through the development of villi and crypts. The adult-type crypt epithelium architecture is present by 30 weeks’ gestational age. Many genes that are necessary for cellular differentiation and migration have been identified ( Table 10.1 , Fig. 10.1 ). The cell types present in the epithelium include enterocytes, goblet cells, enteroendocrine cells, Paneth cells, microfold cells, lymphocytes, and tuft cells ( Table 10.2 ).

Genes involved with differentiation of gastrointestinal epithelium.

From Wyllie R, Hyams J. Pediatric Gastrointestinal and Liver Disease , 5th ed. Philadelphia, PA: Elsevier, 2016.

The intestinal epithelium contains several types of cell junctions that are important for normal functioning. These junctions appear at the 10th week of gestation and continue to develop from that point on. There are four types of junctions, and their primary purpose is to maintain the mechanical and chemical barriers of the GI lumen ( Fig. 10.2 ). Desmosomes are complexes derived from cadherin and are located on the lateral sides of the enterocyte. They function to provide mechanical strength by linking adjacent cells by the cytoskeleton. Adherens junctions are multiprotein complexes that assist with epithelial cohesion, polarity, and cell migration. Gap junctions primarily allow for intercellular chemical communication via molecules, ions, and electrical impulses. Finally, tight junctions are composed of nearly 40 different proteins and are quite possibly the most important type of junction. The tight junction protein components form a continuous ribbon around the cells near the borders of the lateral and apical membranes and serve as a barrier between the GI lumen and the subepithelial environment by preventing the passage of a certain luminal antigens, microorganisms, and toxins. The intercellular components also interact with different scaffold proteins, adapter proteins, and signaling complexes that regulate the cytoskeleton and the polarity of the cell and are involved in cell signaling and trafficking and serve to provide selective paracellular transport of solutes. These tight junctions are dynamic and can adapt to different physiochemical properties in the intestinal lumen. Disruption in the integrity of the tight junction has been associated with many disease processes, including inflammatory bowel disease, celiac disease, and type 1 diabetes.

There are four types of intercellular junctions found in the gastrointestinal epithelium, including desmosomes, adherens junctions, gap junctions, and tight junctions.

From Neunlist M, Van Landeghem L, Mahe MM, et al. The digestive neuronal-glial-epithelial unit: a new actor in gut health and disease. Nat Rev Gastroenterol Hepatol. 2013;10:90–100.

Microbiome and Diversity

Although the development of the microbiome has been recognized as critical to the normal development of the GI tract, it has only recently been noted that colonization may begin before birth. The microbiome continues to expand, and does so shortly after birth through the first 2 years of life. Typical progression begins with colonization of primarily facultative anaerobes, such as Streptococcus , Enterobacteriaceae, and Staphylococcus species within the hours following birth; this quickly expands to include Bifidobacterium and Lactobacillus within the first 48 hours of life. By age 3 months, regionalization of different bacterial species is noted with Fusobacterium and Eubacterium colonizing the colon and Enterobacteriaceae, Streptococcus , and Clostridium found in the ileum ( Fig. 10.3 ). Despite the presence of tight junctions, bacteria and their metabolic byproducts penetrate the submucosa; the interaction between the microbiome and the gut-associated lymphoid tissue (GALT) serves to shape the development of the gut mucosal immune system. This coevolution during postnatal life allows the host and the intestinal microbiome to coexist as symbionts, without limiting the immune response to pathogens. Failure to maintain this symbiotic relationship has been associated with diseases, including obesity, inflammatory bowel disease, atopic conditions, celiac disease, and necrotizing enterocolitis (NEC) in neonates.

The neonatal microbiome evolves over the course of the first 1 to 2 years of life. There are several major shifts in the gut flora, at birth, with introduction of enteral feeds (breast milk, formula), and later with diversification of the diet. The microbiota is comparable to the adult flora profile by 2 years of life.

From Wyllie R, Hyams J. Pediatric Gastrointestinal and Liver Disease, 5th ed. Philadelphia, PA: Elsevier, 2016; and Langhendries JP. Early bacterial colonisation of the intestine: why it matters. Arch Pediatr . 2006;13:1526–34.

Special Neonatal Challenges

In the neonatal population, a variety of environmental factors, such as mode of delivery, diet, and gestational age, can result in altered microbial establishment within the GI tract ( Fig. 10.4 ). Preterm neonates are more at risk for an altered microbiome because of their medicalized environment; these infants are exposed to pathogenic bacteria more frequently as a result of prolonged hospitalization within the neonatal intensive care unit (NICU). In addition, certain therapies, such as antibiotics, compound the issue by reducing species diversity so that preterm infants are often colonized by a less diverse quotient of bacteria. These environmental challenges often produce a microbiome with less beneficial bacteria (bifidobacteria, lactobacilli) and larger populations of potential pathogens ( Klebsiella , enterococci, staphylococci, Bacteroides , and Enterobacteria ). Clinically, this may have both short- and long-term effects; a growing number of studies have correlated prolonged courses of antibiotic therapy with increased incidence of NEC, sepsis, and death.

The preterm neonate has many unique exposures that serve to alter the gastrointestinal microbiome.

In addition to environmental challenges, the preterm neonate has less efficient immune function and abnormal mucosal barriers within the gut that allow for translocation of pathogenic bacteria and initiation of a systemic inflammatory response. Under normal circumstances, the intestinal epithelium serves a challenging role of facilitating transport of nutrients while maintaining a barrier against microorganisms. The physical barrier is composed of the epithelial cells and their tight junctions, as previously described. The intestinal epithelial cells are active members of this barrier; Paneth cells respond to certain signals and respond with antimicrobial peptides. Intestinal epithelial cells also express certain receptors that allow recognition of ligands produced by both commensal and pathogenic bacteria; the recognition of these ligands triggers a cytokine interaction between the intraepithelial lymphocyte and the underlying GALT. The immune response of the GI mucosa comprises both adaptive immune cells, such as effector T and B lymphocytes found in the GALT, as well as innate cells, such as dendritic cells, monocytes/macrophages, and the recently identified heterogeneous group of innate lymphoid cells. The preterm neonate has qualitatively or quantitatively less of these immune cells present.

There are many signaling pathways between the involved cells types; intestinal signaling through Toll-like receptors (TLRs) has been particularly well studied. Rakoff-Nahoum et al. were the first to demonstrate that intestinal signaling through TLRs stimulated by the intestinal microbiome is necessary for homeostasis. The intestinal immune cells must find a balance between mounting appropriate defenses against pathogenic organisms and developing tolerance toward commensal organisms. Excessive activation of the immune response would cause excessive production of inflammatory cytokines, resulting in local cell death and an inflammatory response. The TLR helps maintain balance through cell signaling in response to byproducts, such as lipopolysaccharides (LPSs) produced by commensal bacteria; this pathway ultimately enhances the ability of the epithelial cells to withstand chemical and inflammatory injuries. Significant differences have been identified in the degree of TLR function and TLR subtype activity in preterm infants and full-term infants. TLR4 activity has been noted to be highest in the preterm infant. This pathway is associated with epithelial apoptosis, mucosal barrier disruption, bacterial translocation, and reduced ability to recover from injury ( Fig. 10.5 ). Paneth cells also serve a role in tolerance of intestinal bacteria by producing antimicrobial proteins and peptides; cathelicidin (LL-37) is one such amyloid precursor protein, which functions to decrease inflammatory cytokine response to LPSs. Preterm infants appear to have a shift in balance, from an anti-inflammatory cytokine and protein environment to one that is more proinflammatory. Preterm infants have a higher expression of TLR4 activity, as well as interleukin-8 (IL-8) and IL-1. Protective LL-37 levels have been noted to drop off around 2 weeks after birth in mice models. Prenatal steroids increase intestinal maturation and reduce IL-8 production (via reduction in nuclear factor-κB nuclear translocation), which may help to explain, in part, why this prenatal therapy is associated with reduction in NEC. Overall, the immature innate immune system of the preterm neonate is often insufficient to adequately protect against the numerous challenges faced by it.

Toll-like receptor (TLR)-4 activity is high in preterm neonates and is associated with activation of the inflammatory response with epithelial apoptosis, mucosal barrier disruption, bacterial translocation, and reduced ability to recover.

From Thomson CA, McColl A, Cavanagh J, Graham GJ. Peripheral inflammation is associated with remote global gene expression changes in the brain. J Neuroinflamm. 2014;11:73.

There are several basic physiologic mechanisms that are not fully functional in the preterm population, compared with those in full-term infants; these include peristalsis, gastric acidity, presence of proteolytic activity, intestinal mucus, cell-surface glycoconjugates, and intercellular tight junctions. There are immunologic differences, including decreased secretory immunoglobulin A (IgA) production and less antimicrobial peptides (specifically defensin) secretion by Paneth cells. These anatomic, physiologic, and immunologic discrepancies compound each other, resulting in a less capable intestinal mucosa that is more susceptible to damage, less able to heal itself, and more at risk for bacterial translocation. Clinically, this is thought to contribute to bacterial overgrowth, dysbiosis, and NEC. In summary, the initial development of a symbiotic relationship between the intestinal mucosa and the microbiome is critical to a neonate’s health. In preterm infants, several mechanisms, such as innate immune function and mucosal integrity, function either suboptimally or with a proinflammatory shift compared with their full-term counterparts, which increases the risk of sepsis, systemic inflammatory responses, and NEC.

Gastrointestinal Impact on Systemic Inflammation

The effects of environmental stressors on the gut epithelium and subsequent alterations in the intestinal microbiome are not solely confined to the GI tract. Instead, there is a dynamic relationship between the GI integrity and systemic inflammation. When the epithelial tight junctions are compromised, translocation of bacteria occurs; this can trigger a gut-derived inflammatory response, with production of toxic mediators that drive systemic inflammation, which, then, feeds back and promotes increased intestinal permeability and further local immune activation. This cyclic inflammatory cascade ultimately can drive the body to systemic inflammatory response syndrome (SIRS), sepsis, septic shock with multiorgan failure, and even death. Several mechanisms are thought to contribute to this gut–body inflammatory relationship ( Fig. 10.6 ).

There is a reciprocal relationship between local gastrointestinal (GI) and systemic inflammation, in which the GI tract may serve as an initiating trigger for systemic inflammation and vice versa.

Systemic stress can serve as an inciting factor for GI derangements, such as denudation of villi, impairments in the gut barrier, and translocation of luminal contents. Major shifts to pathogenic species have coincided with reduced microbial diversity that occurs in neonates with SIRS and sepsis. Commensal microflora are vital to the digestion of dietary substrates, prevent colonization of pathogens, encourage enterocyte differentiation and proliferation, and help prime the mucosal and systemic immune systems. They are also involved with fermentation of complex carbohydrates, which results in the production of short-chain fatty acids; this has demonstrated beneficial effects on immune cells and also mediates anti-inflammatory effects through G-protein coupled receptors 41 and 43 (GPR41, GPR43). One could conclude that potential shifts in the microflora may alter this normal homeostatic function. Although there are, as yet, no routinely used means to measure bowel function in clinical practice, one potential means to do so is citrulline. Plasma citrulline levels are representative of enterocyte function and mass, and decreased levels may be associated with loss of gut barrier integrity. Low citrulline levels in adult patients have been associated with elevated C-reactive protein levels, indicating systemic inflammation, as well as with higher mortality.

Although inflammation may begin with an insult to the intestine, resulting from microbiota changes or ischemic injury, it is not necessarily contained there. The portal vein is composed of tributaries that drain the majority of GI blood products from the lower third of the esophagus to halfway down the anal canal and to the spleen, pancreas, and gallbladder. The portal vein flows into the liver, providing approximately 70% of the organ’s blood flow—which makes the liver an important filter for clearing systemic bacterial infections derived from gut barrier dysfunction and for maintaining gut homeostasis. The hepatic sinusoids are lined with Kupffer cells, which are macrophages that are often the first immune cells to encounter pathogens that translocated from the gut lumen. Dysbiosis of the microbiome has been found to trigger local inflammation within the liver and in patients with underlying liver disease (nonalcoholic fatty liver disease) may prompt progression from moderate disease to steatohepatitis. A study in rat models noted that microbiota-dependent activation of the chemokine receptor (CX3CR1) in intestinal macrophages (which affects guts barrier integrity) regulates progression to steatohepatitis. This is a good example of how damage to the GI barrier subsequently leads to inflammation within the liver, which, in turn, can result in circulatory changes that then worsen intestinal damage. The relationship between gut microflora and the immune response within the liver has been further explored in recent studies; Balmer et al. found that although the liver remains sterile during periods of intestinal health, during times of translocation, the liver becomes a “secondary firewall” for mesenteric circulation. Microbial products, such as LPS or bacterial DNA, can also breach the intestine and affect the liver; these events have been associated with activation of the inflammasome complex and systemic inflammatory responses. The recognition of microbial antigens activating TLRs on hepatic cells (Kupffer cells, stellate cells, hepatocytes, lymphocytes, and endothelial cells) results in production of proinflammatory cytokines. As in the intestines, the TLR plays a role in liver homeostasis. Although stimulation of TLRs on Kupffer cells is generally proinflammatory, continuous low levels of LPS stimulation, in fact, induces LPS tolerance in the liver and secretion of antiinflammatory cytokine IL-10. This interplay between the GI microbiota and the liver may serve as one pathway and explains GI changes being a trigger for systemic inflammatory processes.

The lymphatic system offers another venue through which the gut and systemic circulation are connected. Mesenteric lymph containing lipophilic macromolecules drains from the intestinal villi into the mesenteric lymph nodes and ultimately into systemic circulation via the thoracic duct. Within the lymph nodes, antigen presentation occurs and can spur activation of the adaptive immune system. The mesenteric lymph does not go into portal circulation, thus avoiding the “secondary firewall” established by the liver. Lymph may also contain luminal byproducts, such as endotoxins and locally produced cytokines. These cytotoxic factors, in addition to activated immune cells that exit the mesenteric lymph node, are able to merge into systemic circulation, where the pulmonary circulation sees them initially. There have been adult studies correlating this “gut–lung–lymph” axis, with direct toxic pulmonary effects on the pulmonary endothelium and subsequent lung injury and even acute respiratory distress syndrome. Interestingly, in animal models, this has not been demonstrated to be associated to microbial products but, instead, was related to various acute-phase proteins, possibly generated at the intestinal epithelium ( Fig. 10.7 ). Paneth cells are a key source of multiple proinflammatory products that may serve to influence the initiation of a systemic inflammatory response. Paneth cells are found to secrete both α-defensin and phospholipase A2, which is an enzyme that generates lipids mediators, such as prostaglandins ; the levels of both these products have been found to be increased in toxic mesenteric lymph. When activated, they also produce IL-17A, which is a proinflammatory cytokine that serves to activate neutrophils. In mouse models, Vandenbroucke et al. found that mice that are deficient in matrix metalloproteinase 7 (MMP7), which is an enzyme required for posttranslational activation of Paneth cells, were protected against LPS-inducted lethality. A study by Lee et al. found increased levels of IL-17A in the small intestine, liver and plasma after mice were subjected to SMA ischemia (ischemia/reperfusion insults), resulting in small bowel, hepatic, and renal injuries; however, they noted that genetic knockout mice for IL-17A were protected against both intestinal damage and subsequent hepatic and renal effects.

The gastrointestinal tract has several mechanisms in which it may trigger a systemic inflammatory response.

From De Jong PR, Gonzalez-Navajas JM, Jansen NJ. The digestive tract as the origin of systemic inflammation. Crit Care. 2016;20:279.

More data are becoming available from studies on animal models demonstrating the active role that the GI tract plays in systemic inflammation. In the preterm infant, local changes, such as the microbiome alterations caused by medical interventions (e.g., antibiotics), underlying host factors affected by prematurity, and source of nutrition that certainly affects the local GI inflammatory response, are involved. The GI tract itself was once thought only to act as a sieve through which translocation occurs, but current data suggest a much more dynamic relationship. It seems that there is significant interplay through which the GI system serves as an initiation point and a promoter of systemic inflammation during times of stress, even outside of NEC and local bacterial translocation.

Developmental Sequelae of Inflammation

In preterm infants there are unique pathologies, such as chorioamnionitis, sepsis, and NEC, all of which generate significant systemic inflammation. The neonatal mortality following severe inflammatory and infectious insults, such as NEC and septic shock, has been reported to be as high as 30% to 40%. There is often significant neurodevelopmental morbidity in patients who do survive. Multiple studies have described the association between poor neurodevelopmental outcomes in preterm infants surviving after clinical conditions associated with severe neonatal inflammatory insults. Stoll et al. noted an increased risk for impairment in vision, hearing, and mental and psychomotor developmental indices, as well as reduction in brain size, in patients with sepsis and NEC. Preterm infants are more susceptible to ischemia, inflammation, and resultant free radical attacks, which predispose them to loss of premyelinating oligodendrocytes and white matter injury. Sepsis, NEC, and recurrent infection-mediated neurodevelopmental impairment are associated with systemic upregulation of inflammatory cytokines and diffuse activated microgliosis, as well as glutamate receptor–mediated oligodendrocyte injury leading to maturation-dependent cell death and loss of cellular processes (excitotoxicity). Each of these factors contributes to white matter injury seen on brain magnetic resonance imaging. The involvement of many mechanisms in the relationship between long-term neurodevelopmental outcomes and systemic inflammation is an area that requires further research.

Components and Functions of the Gastrointestinal Epithelium

The GI surface is composed of several types of cells. The epithelial layer originates from the endoderm and throughout gestation changes and differentiates through recanalization of the intestinal lumen and, later, through the development of villi and crypts. The adult-type crypt epithelium architecture is present by 30 weeks’ gestational age. Many genes that are necessary for cellular differentiation and migration have been identified ( Table 10.1 , Fig. 10.1 ). The cell types present in the epithelium include enterocytes, goblet cells, enteroendocrine cells, Paneth cells, microfold cells, lymphocytes, and tuft cells ( Table 10.2 ).

Table 10.1

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