Neonatal Gastrointestinal Innate Immunity

After completing this article, readers should be able to: Evidence is accumulating that the defense mechanisms in the newborn that normally maintain a healthy balance in the intestine (both immune and nonimmune) may be inadequate or at least not functioning at levels observed in adults. Infants, particularly undernourished infants, may be at an increased risk for luminal proliferation of and mucosal invasion by microbial pathogens and intact dietary antigens.The gastrointestinal (GI) tract presents the largest surface area of the body to which antigens and microbes are exposed. The intestine, therefore, must have intricate mechanisms to allow entry of nutrients and other beneficial molecules while preventing potentially harmful microbes and other agents from gaining entry into the inner milieu. Several factors lead to a hostile GI environment that predisposes the preterm infant to disease. These include the introduction of feeding tubes into the stomach or more distal intestine, the routine use of broad-spectrum antibiotics that select for resistant pathogens that thrive in the unusual microbial environment of the neonatal intensive care unit, intrinsic immaturities of the infant GI tract, and the lack of adequate nutrition.Intestinal defenses may be broadly divided into immune and nonimmune categories. The nonimmune system sometimes also is referred to as the innate immune system (Fig. 1). Although there is considerable overlap between the immune and nonimmune systems, this review focuses primarily on the innate arm of the intestinal defense system, which constitutes the first line of host defense. The immune defenses refer to specific humoral and cellular immune responses that are activated in response to particular antigens. Each of the components of the innate system acts in concert to maintain mucosal integrity in the intestine.Several studies suggest that the prevalence of type 1 diabetes, asthma, and atopy is lower in children who were breastfed as infants. Human milk protects the intestine from infectious pathogens and intact dietary antigens. Numerous components of human milk are believed to be protective, including immunoglobulins, lactoferrin, lysozyme, glycoconjugates, oligosaccharides, and various types of white blood cells. Antibodies in human milk reflect the antigenic repertoire of the mother’s intestine and respiratory tract. The repertoire of antibacterial, antiviral, antifungal, and antifood antibodies in human milk is considerable. It varies from mother to mother and is determined by the antigens ingested and inhaled. If the mother and infant are colonized by the same bacteria, the mother can produce specific antibodies that can protect the infant through her milk. This has been referred to as an enteromammary circuit (Fig. 2).Intestinal motility can be a critical factor in clearing antigens presented to the intestinal mucosal barrier from the gut lumen. The time available for absorption depends on the transit speed of luminal contents down the bowel. Migratory motor complexes act as “housekeepers” to propel luminal contents caudally along the length of the small intestine. Motility disorders alter normal peristaltic activity and result in overgrowth of anaerobic bacteria in the small intestine, with malabsorption of dietary nutrients. In postnatal life, motility patterns change with development and may not reach a mature pattern (comparable to that that of the term infant) until 34 to 35 weeks postgestation.Normal gastric acid and pancreaticobiliary secretions decrease the load of viable microorganisms as well as intact dietary protein antigens that reach the small intestine. Pancreatic insufficiency in the preterm infant can last through the first year of life. Lack of stimulation of gastric acid and pancreaticobiliary secretions can affect the intestine adversely by allowing a greater bacterial or antigenic load (see the article on the neonatal gastrointestinal tract in this issue of NeoReviews). Enzymatic and chemical proteolysis destroy the structure of antigens, thereby destroying the epitopes for immunologic recognition. Many very low-birthweight infants are not provided nutrients by the enteral route for the first several days after birth and consequently have minimal intestinal protease and acid stimulation. These same infants frequently are given histamine2 (H2) blocking agents to decrease gastric acid secretion. Accordingly, the breakdown of intact protein molecules, bacteria, and other microbes may be less. The detrimental effect of this practice is evidenced by the greater incidence of hospital-acquired sepsis seen in infants who receive H2 blocking agents.Intestinal mucus plays a significant role in intestinal defense (Fig. 3). Mucus is a complex gel that covers the surface of the villous epithelium. It is comprised primarily of water and electrolytes, but it also contains mucins, glycoproteins, immunoglobulins, glycolipids, and albumin. Bioactive factors such as lactoferrin, lysozyme, and glycolipids from human milk also may be contained within the surface mucus gel. Mucin prevents direct translation of hypo- and hyperosmolar forces to the mucosal surface. It also surrounds foreign antigens and assists in their propulsion distally within the lumen. Mucus is secreted into the lumen of the intestine from storage vacuoles in goblet cells that are interspersed among enterocytes along the villus-to-crypt axis. Mucous gels containing foreign dietary antigens and pathogenic bacteria are sufficiently stable to keep their contents trapped.The intestine is capable of responding to a threatening insult by the rapid outpouring of mucin. The mechanism of this response not is well understood. Mucin molecules are a heterogeneous group, and marked changes in their composition occur during development. For example, mucin from the small intestine of newborn rats contains more protein than mucin from adult rat intestine. Carbohydrate composition also changes; the newborn mucin has less fucose and N-acetyl-cysteine than adult mucin. Levels of mucin in the small intestine decrease in experimental protein-calorie undernutrition. Lower amounts of mucin caused by malnutrition could affect the normal protective function of mucin, particularly in the perinatal period. Mucin also has its greatest effect on reducing diffusion of large molecules when compared with small molecules. This effect limits the absorption of antigens rather than nutrient molecules, which are smaller.The lining epithelium of the primitive intestine initially is stratified, but it becomes a single layer of columnar cells at the end of the first trimester. The intestinal epithelial enterocyte is derived from the same undifferentiated stem cell that gives rise to columnar mucous neck, goblet, enteroendocrine, and probably M cells in the intestine (Fig. 4). Interspersed between intestinal epithelial enterocytes are blood-derived intraepithelial lymphocytes (IELs). This unique T-cell population is localized to the basolateral side of the epithelial layer, where the cells are exposed to a varied range of food and microbial antigens. These cells proliferate poorly. They are cytotoxic and capable of producing cytokines. One well-established function of the IEL is protection of the host from invasion by microorganisms that enter through the GI tract. Goblet cells are found throughout the small and large intestine. They secrete mucus, which is very important in the nonspecific protection of the gut lining, as previously described. Rats that are specifically immunized exhibit goblet cell hyperplasia. This hyperplasia is dependent on helper T cells, which are known to secrete proliferation and differentiation factors classified as lymphokines. When specifically stimulated, it is likely that goblet cells respond to one or more of these lymphokinesSpecial tight junctions and interdigitations join the intestinal epithelial cells. The tight junction (zonula occludens) is the most apical component of the junctional complex and serves as a permeability barrier between the external and internal milieus of the body (Fig. 5). The tight junction completely encircles the apical end of absorptive cells as a beltlike band. Several transmembrane proteins and proteins on the cytosolic leaflet are a part of the tight junction. Interactions of some of these proteins with the actin cytoskeleton are a major determinant of tight junction structure and play a role in the regulation of tight junction assembly. Just below the tight junction is the adhesion junction (zonula adherens), which is connected to actin filaments in the cytoplasm and anchors each cell to adjacent cells. Below this, the desmosome (macula adherens) functions in a similar manner to the zonula adherens. The relationship between density of zonula occluden strands and epithelial resistance has been demonstrated in cell culture studies. These junctions allow for physiologic passage of fluids, electrolytes, and small peptides up to 11 amino acids long, but not for larger proteins under normal physiologic conditions. On the other hand, pathologic insults may alter these junctions so they allow unregulated passage of particles. These intercellular junctions are exquisitely sensitive to modulation by small molecules, such as the short-chain fatty acids butyrate and glutamine.M cells in the intestine do not have well-developed microvilli and allow macromolecular transport; the absorptive enterocyte has better developed microvilli (Fig. 6). M cells occur only in the follicle-associated epithelia that overlie organized mucosa-associated lymphoid tissues. They are specialized for delivering foreign antigens and microorganisms to organized lymphoid tissues within the mucosa of the small and large intestines. This has advantages and disadvantages in that certain pathogens exploit exactly those features of M cells that are intended to promote uptake for the purpose of immunologic sampling.It is unclear whether the infant can respond to injury to the same extent as the adult. In nonhuman mammals, the intestinal epithelial turnover is much slower in the infant (4 to 5 d) than in the adult (2 d). If the turnover in human neonates is similarly longer than the adult, this may be significant because regeneration of injured mucosa in the infant will be much slower than in the adult.Microvilli constitute a significant barrier because of their size and charge. The distance between microvilli in children is estimated to be about 25 mcm. Because microvilli are negatively charged, large macromolecules and charged particles may have difficulty penetrating this barrier. The sites of invagination of plasma membrane are between the microvilli. It is likely that antigens have to pass this microvillus “barrier” to enter the cell. Thus, the closer the microvilli are to each other, the less likely that large antigens will pass. Infections, lack of appropriate nutrition, or other factors that may alter the microvillus barrier could provide a greater potential for antigenic entry into the epithelium.Compositional changes can alter the fluidity of the fluid-mosaic structure of the microvillus membrane. Such changes are associated with developmental maturation and hormone concentration. These correlate with changes in lectin binding and may translate to alterations that increase the potential for adhesion of microorgansims or permeability.The rate of vesicular passage from the apical to the basolateral membrane is important. Endosomes with lysosomally derived enzymes (eg, proteases, cathepsin, acid phosphatases) or endocytic vesicles destroy some of the macromolecular biologic activity of proteins and fluctuations in other antigens. However, they may not completely digest the protein molecule, and the final steps in digestion of peptides may be by peptidases. Whether these enzymatic activities are fully developed in preterm infants is not clear.Follicular dendritic cells are important nonphagocytic antigen-presenting cells. They are present in lymphoid follicles and are essential in presenting antigen to CD 4+ T cells as well as B cells. They play a central role in the maintenance of local immunologic homeostasis. Peyer patches are aggregations of lymphoid tissue in the lamina propria and submucosa of the GI tract opposite the mesentery. These develop throughout the small intestine, but become more prominent in the adult in the terminal ileum. Peyer patches become evident at about 19 weeks of gestation. Their numbers increase from 24 to 40 weeks of development within the ileum and jejunum. M cells occur in the follicle-associated epithelium over the dome area of Peyer patches. This area contains few goblet cells and because of this, luminal antigens have no mucus layer to traverse before contacting cell surfaces. M cells transport large molecules, certain bacteria, and viruses. Mast cells release mediators that are very important in GI tract immunity. They bind immunoglobulin E (IgE) via their Fc epsilon receptors and subsequently release histamine and serotonin, which stimulates mucus production by goblet cells and increases enterocyte permeability, chemotaxis of granulocytes, contraction of smooth muscle cells, and lymphocyte function.Numerous innate humoral factors are secreted onto mucosal surfaces by exocrine glands, and they can carry out their protective functions in the absence of specific antibodies. They protect the mucosa against pathogens and other insults before the immune system has had time to mount specific responses. They are present as a family in mucosal secretions and act synergistically in performing their important function. The factors include lactoferrin, lysozyme, peroxides, trefoil peptides, phagocyte cells, and other bioactive factors. A complete review of all innate humoral factors is beyond the scope of this article, but it is instructional to address lactoferrin.Lactoferrin is a multifunctional protein produced in neutrophils and stored in the iron-depleted state. Human lactoferrin consists of 703 amino acid residues. The protein is folded into two globular lobes, forming a three-dimensional structure that binds up to two iron atoms. The anti-infective properties of lactoferrin include bacteriostatic, bactericidal, antifungal, and antiviral effects.Infants fed human milk are more resistant to intestinal infections than those fed formula. Lactoferrin is present in relatively high concentrations in human milk, suggesting a protective role that is conferred to the developing infant. Some ingested lactoferrin can survive digestion by proteolytic enzymes in the gut. Lactoferricin, the pepsin-cleavage product of lactoferrin, also has potent antimicrobial activity and may contribute to biologic defense in the infant’s gut.The mucosal immune system is the first line of defense for more than 95% of infectious bacterial and viral diseases. Integral to the system is a class of compounds called the cytokines. These soluble messenger molecules interact with cells and receptors to orchestrate the production of mucosal antibody and cell-mediated responses. Among the characteristics of cytokines, which are polypeptides or glycoproteins that have a molecular weight generally less than 30 kDa, are: The cytokines play many roles, including chemotaxis of neutrophils, promotion of IgA expression, epithelial cell proliferation, differentiation, and restitution after mucosal injury.The role of cytokines produced by the intestinal epithelial cell (IEC) is just beginning to be elucidated. Interleukin-6 (IL-6) has important proinflammatory functions, such as inducing secretion of acute-phase proteins by the liver and enhancing T-cell proliferation and B-cell antibody secretion. Epithelial cells located at the base of the crypts, called Paneth cells, have been found to express mRNA for the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha). IECs also secrete various types of chemokines, a group of small proteins produced in a variety of cell types in response to inflammatory stimuli. The first chemokine found to be produced by IEC was IL-8, which was induced by TNF-alpha. The peak stimulation occurred at about 4 hours. IL-8 now is known to be a potent inducer of leukocytic infiltration into tissues.Although one of the most obvious functions of the IEC is to maintain a functional barrier to intestinal luminal contents, the cytokine-related inflammatory response drastically alters this barrier function. Interferon gamma, another proinflammatory cytokine, can reduce the epithelial barrier function between cells grown in culture. Transforming growth factor-beta counteracts this decrease, which is consistent with its other known functions, primarily cell restitution and wound healing.The epithelial cell is an important participant in the immune and inflammatory response at mucosal surfaces. This cell layer may be an excellent focus for therapeutic intervention or prevention to control mucosal inflammatory responses. This should not be limited to anticytokine therapy. Immnonutrients, such as amino acids and long-chain lipids (described further in the article on immunonutrients in this issue of NeoReviews) also could play a role in modulating intestinal epithelial cytokine secretion directly or by altering the surface properties of the mucosa. This could prevent interaction of antigens with the mucosal surface or their translocation by paracellular or transcellular routes, which in turn can prevent the inflammatory response in submucosal cells.Previous data suggest that both animals and humans who have a propensity to develop type 1 diabetes have an abnormally functioning intestinal barrier. For example, increased permeability to otherwise nonabsorbable sugars has been noted in diabetes-prone rats (Fig. 7) before the onset of diabetes. This allows for potentially diabetogenic antigens to traverse a barrier when the individual may be particularly susceptible, such as during times of GI infection. For example, infectious processes such as rotavirus infection frequently precede the onset of type 1 diabetes. These conditions result in alterations of the intestinal barrier that allow for increased “leakiness” and penetration of usually nonabsorbed substances (Fig. 8). The frequent association of type 1 diabetes with celiac disease, which also exhibits altered intestinal permeability, raises suspicion about increased intestinal permeability playing a role in the pathogenesis of type 1 diabetes. Other studies have suggested that nutritional strategies that present less immunogenic materials (protein hydrolysates) to the intestine delay the onset of diabetes and increase survival (Fig. 9). This area is fertile for further investigation and may relate to other autoimmune disorders, such as atopy and asthma.The intestine, which has the largest interface between the external environment and the internal milieu, harbors a highly complex system that allows for assimilation of certain molecules and exclusion of others. The first line of defense is the innate immune system, which works in association with the adaptive immune system. We are just beginning to understand the intricacies of these systems and how they relate to health and disease.