REVIEWS. The impact of perinatal immune development on mucosal homeostasis and chronic inflammation

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1 The impact of perinatal immune development on mucosal homeostasis and chronic inflammation Harald Renz 1, Per Brandtzaeg 2 and Mathias Hornef 3 Abstract The mucosal surfaces of the gut and airways have important barrier functions and regulate the induction of immunological tolerance. The rapidly increasing incidence of chronic inflammatory disorders of these surfaces, such as inflammatory bowel disease and asthma, indicates that the immune functions of these mucosae are becoming disrupted in humans. Recent data indicate that events in prenatal and neonatal life orchestrate mucosal homeostasis. Several environmental factors promote the perinatal programming of the immune system, including colonization of the gut and airways by commensal microorganisms. These complex microbial host interactions operate in a delicate temporal and spatial manner and have an important role in the induction of homeostatic mechanisms. 1 Institute of Laboratory Medicine and Pathobiochemistry, Molecular Diagnostics, Philipps University Marburg, Medical Faculty, Baldingerstrasse, Marburg, Germany. 2 Laboratory for Immunohistochemistry and Immunopathology (LIIPAT), Centre for Immune Regulation (CIR), University of Oslo, and Department of Pathology, Oslo University Hospital, Rikshospitalet, Oslo, Norway. 3 Institute for Medical Microbiology and Hospital Epidemiology, Hannover Medical School, Hannover, Germany. Correspondence to H.R. renzh@med.uni-marburg.de doi: /nri3112 Published online 9 December 2011 The incidence and prevalence of chronic inflammatory diseases have increased markedly since the end of the Second World War, in line with the pattern of increasing urbanization and industrialization. This is particularly evident for inflammatory disorders that develop in the mucosal tissues of the airways and the gut, such as asthma and chronic inflammatory bowl disease (IBD) 1. The hygiene hypothesis was introduced to explain the increased incidence of these diseases in affluent societies 2,3. According to this theory, modern hygiene, and dietary and medical practices affect the composition of the gut microbiota and limit exposure of infants to pathogens. This change in the microbiota, in combination with genetic and epigenetic factors, influences not only the epithelial mucosal barrier but also perinatal maturation of the immune system, thus leading to disease susceptibility. Mucosal tissue homeostasis results from the perinatal establishment of mucosally induced immune tolerance, which has been extensively studied in terms of immunological hyporesponsiveness to ingested innocuous antigens (known as oral tolerance) and to components of the indigenous gut microbiota. Similar tolerogenic mechanisms can be induced through the respiratory mucosa. Perinatal defects in the induction of mucosal tolerance are associated with the later development of allergies, autoimmune diseases (such as rheumatoid arthritis, type 1 diabetes and systemic lupus erythematosus) 4 6 and chronic inflammation of the gut and respiratory mucosae. In both the gut and the airways, mucosal tolerance is regulated by a set of signals provided by innate immune cells that shape adaptive immune responses. The mucosal epithelium controls this regulatory immune network through its barrier function, cell contact-mediated signals and the production of cytokines. It is now well recognized that environmental factors, either directly or indirectly, have a decisive role in perinatal maturation of the mucosal immune system. The colonization of mucosal surfaces by commensal microorganisms is of eminent importance 7,8. Although current research focuses mainly on the intestinal microbiome, the functional relevance of the respiratory microbiome is also emerging 9,10. In addition, perinatal exposure to cigarette smoke, environmental microorganisms and dietary constituents has a marked effect on the early programming of the innate and adaptive immune systems through the stimulation of pattern recognition receptors (PRRs) and by modifying cellular interactions; thus, such exposure directly influences disease development. Indeed, identified susceptibility genes for both respiratory and enteric chronic inflammatory diseases are associated with epithelial barrier integrity, innate immune recognition and adaptive immune stimulation NATURE REVIEWS IMMUNOLOGY VOLUME 12 JANUARY

2 Oral tolerance This form of tolerance is established through the intestinal mucosa to avoid local and systemic hypersensitivity to innocuous antigens that have breached the epithelial barrier. It is mediated mainly through the induction of regulatory T cells but probably also through other mechanisms, such as T cell anergy and clonal deletion. Crypt villus architecture Crypts are glandular invaginations of the intestinal epithelium. Paneth cells localize to the base of crypts in the small intestine and secrete antimicrobial peptides. Intestinal stem cells in crypts divide continuously and provide rapid epithelial cell renewal. In the small intestine, villi are finger-like protrusions into the gut lumen, which increase the absorptive surface of the gut epithelium. Villi mainly consist of mature, absorptive enterocytes, but also contain mucus-secreting goblet cells. Cathelicidin Cathelicidins are a large family of antimicrobial peptides in ruminants. In mice and humans, only one cathelicidin CRAMP or LL37, respectively is expressed. The genes encoding CRAMP and LL37 are expressed by most epithelial cells, as well as granulocytes, and their expression is regulated at the level of transcription. They encode a highly conserved cathelin sequence of 12 kda at the amino terminus, followed by the mature antimicrobial peptide, which requires enzymatic cleavage to become active. Defensins A class of antimicrobial peptides with broad antibacterial, antifungal and, in part, antiviral activity. α defensins are produced constitutively by small intestinal Paneth cells and, in humans, also by neutrophils, whereas many β defensins are transcriptionally regulated and expressed by most epithelial cells. This Review focuses on disorders of the mucosae but does not present a complete picture of the many factors that contribute to inflammatory diseases of the gut and airways. Instead, we highlight perinatal variables that influence mucosal barrier function and host defence and that when dysregulated can contribute to the patho genesis of chronic inflammatory disorders of the respiratory and intestinal tracts. Perinatal development of the gut mucosa The intestinal mucosal barrier. The level of maturity of the neonatal gut varies between species, depending on the length of the gestation period. The small intestinal mucosa of human newborns has a mature crypt villus architecture, with continuous stem cell proliferation, and epithelial cell migration and differentiation. In mice, small intestinal crypts only develop days after birth and this is accompanied by increased epithelial cell renewal and the transcriptional reprogramming of enterocytes, which involves changes in the expression of genes involved in nutrient metabolism, nutrient transport and cell differentiation 14,15. Also, the enteric spectrum of antimicrobial peptides changes significantly during neonatal development in mice. During the first two weeks of postnatal life, when mature crypt-based Paneth cells are absent 16,17, the mouse intestinal epithelium expresses cathelicidin-related antimicrobial peptide (CRAMP) 18. CRAMP expression decreases with weaning, at the same time that Paneth cells start to produce defensins (FIG. 1). Whereas defensin secretion by mature Paneth cells does not depend on bacterial colonization of the gut 19, expression of another antimicrobial peptide by epithelial cells of the adult intestine the C type lectin regenerating isletderived protein 3γ (REG3γ) requires bacterial colonization of the intestine and is supported by interleukin 22 (IL 22)-producing RORγt + NKp46 + lymphocytes 20,21. Of note, changes in the composition of antimicrobial peptides and in antibacterial activity during the early postnatal period have also been noted in the intestinal lumen of human neonates 22. Thus, significant alterations of the antimicrobial peptide repertoire of the intestinal mucosa occur during the early postnatal period. As enteric antimicrobial peptide synthesis has been associated with changes in the composition of the microbiota and chronic mucosal inflammation, these processes during the postnatal period might significantly affect susceptibility to inflammatory disease later in life 23 (FIG. 1). Colonization of the gut mucosa by commensals. Birth marks the transition from a sterile fetal environment to one that is rich in microorganisms and nutritional or other exogenous substrates 24,25. The first microbial encounter of a neonate seems to determine the early postnatal intestinal microbiota: after natural birth, the microbiota composition resembles that of the maternal vaginal or gut microbiota, whereas after Caesarean section, the intestinal microbiota of the infant includes a large number of environmental bacteria 25. With weaning, an increasingly diverse microbiota is established that is highly individual and remains relatively stable throughout life 26,27. Perinatal microbiota epithelial crosstalk. Exposure of mouse pups during or shortly after natural birth to lipopoly saccharide (LPS) from Gram-negative bacteria induces a transient transcriptional activation of the small intestinal epithelium with upregulated expression of microrna 146a (mir 146a). Increased levels of mir 146a in intestinal epithelial cells cause translational repression of the Toll-like receptor (TLR) signalling molecule IL 1 associated kinase 1 (IRAK1). This, together with the proteasomal degradation of IRAK1, contributes to innate immune tolerance by inhibiting TLR signalling 28. Other mechanisms that help to prevent inappropriate immune stimulation by TLR agonists during postnatal colonization of the neonatal intestine include the downregulation of expression of TLR4 in the intestinal epithelium, expression of which is high in late fetal life of mice but decreases at birth 29 (FIG. 1). Conversely, epithelial expression of the nuclear factor-κb inhibitor IκBα steadily increases during the postnatal period 30. The combination of decreasing levels of TLR4 and increasing levels of IκBα expression by gut epithelial cells effectively increases the threshold of immune activation in the gut epithelium (FIG. 1). Interestingly, decreased IRAK1 protein expression in mouse neonatal epithelium requires continuous TLR signalling. This facilitates prolonged upregulation of mir 146a expression and simultaneously induces sustained expression of genes supporting cell maturation, survival and nutrient absorption 31. Innate immune signalling by epithelial cells seems to be essential for immune tolerance, as lack of the pro-inflammatory signalling molecule transforming growth factor-β (TGFβ)-activated kinase 1 (TAK1) specifically in the murine intestinal epithelium leads to early inflammation, tissue damage and postnatal mortality 32. Thus, although inappropriate stimulation of the neonatal innate immune system by the microbiota must be prevented, controlled innate immune activation significantly contributes to nutrient absorption, angiogenesis, epithelial cell differentiation and barrier fortification 33,34. Despite the decreased sensitivity of epithelial cells to TLR stimulation in murine neonates, other innate immune signalling pathways remain fully functional. For example, rotavirus infection of the intestinal epithelium in neonatal mice is efficiently sensed by the helicases retinoic acidinducible gene I (RIG I) and melanoma differentiationassociated gene 5 (MDA5) 35,36. So, when confronted with the emergence of the intestinal microbiota, the neonatal gut epithelium seems to adjust its sensitivity to microorganisms and modify its signalling pathways after initial stimulation while maintaining antiviral host defences. Perinatal maturation of the intestinal immune system. It is well known that both endogenous and exogenous factors drive the development and maturation of the intestinal immune system (FIG. 1). In mice and humans, the formation of secondary lymphoid structures such as Peyer s patches and mesenteric lymph nodes (MLNs) occurs before birth, but their size and the development of germinal centres depend on the postnatal microbial colonization of the gut Also, cryptopatches and isolated lymphoid follicles are seen only postnatally in mice JANUARY 2012 VOLUME 12

3 CX 3 CR1 + macrophage Maternal SIgA CRAMP Commensal bacterium M cell Bacterial colonization T Reg cell SIgA CD103 + DC Increased mucus secretion Villus T cell Dendritic cell B cell Peyer s patch Mesenteric lymph node IgA-producing plasma cell LTi IEL Cryptopatch ILF Paneth cell maturation Paneth cell Increased epithelial cell proliferation Crypt ILF Defensin Mesenteric lymph node T Reg cell Peyer s patch CCR9 + α4β7 + Epithelial cell proliferation Production of antimicrobial peptides Commensal density and complexity CRAMP Paneth cell-derived peptides and REG3γ Innate immune sensing TLR4 expression IκBα expression No TLR stimulation Tolerance to TLR stimulation Reactivity to TLR stimulation Level of IgAproducing plasma cells Lymphocyte homing and differentiation Prenatal intestine Neonatal intestine Adult intestine Birth Weaning Figure 1 Postnatal development and maturation of the intestinal mucosal barrier and immune system. Both developmental and environmental signals drive significant changes of the intestinal epithelium during Nature the Reviews postnatal Immunology period in mice and accompany the establishment of an increasingly complex and dense gut microbiota. The neonatal intestinal mucosa is characterized by low levels of epithelial cell proliferation, the absence of crypts and crypt-based Paneth cells, and expression of cathelicidin-related antimicrobial peptide (CRAMP); by contrast, the formation of intestinal crypts late during the second week after birth initiates increased proliferation and rapid epithelial cell renewal, the generation of α-defensin-producing Paneth cells and the upregulation of expression of the antibacterial C-type lectin regenerating islet-derived protein 3γ (REG3γ). A decrease in the level of expression of Toll-like receptor 4 (TLR4) by epithelial cells before birth and a steady increase in the RORγt intestinal expression level of the nuclear factor-κb inhibitor IκBα during the postnatal period decrease the responsiveness to + NKp46 + lymphocytes bacterial lipopolysaccharide and other pro-inflammatory stimuli. Simultaneously, the acquisition of epithelial TLR tolerance A population of innate creates a neonatal period of decreased innate immune responsiveness. Note that the human small intestinal epithelium at lymphoid cells in the intestinal birth has a much more mature phenotype than in mice. The secondary lymphoid structures of Peyer s patches and lymph lamina propria that expresses nodes are generated before birth in mice and humans but mature during the postnatal period. By contrast, cryptopatches and the transcription factor retinoic isolated lymphoid follicles (ILFs) are formed after birth in mice. Specialized epithelial cells, known as M cells, reside above ILFs acid receptor-related orphan and Peyer s patches and facilitate antigen transport from the lumen to the underlying lymphoid cells. Simultaneously, innate receptor-γt (RORγt) and the lymphocytes (such as lymphoid tissue inducer (LTi) cells) and T cells leave the liver and thymus, respectively, and colonize the natural killer cell marker enteric mucosal tissue, including the epithelium. Intraepithelial lymphocytes (IELs) reside in close proximity to the epithelium. NKp46. RORγt + NKp46 + lymphocytes support the Also, increasing numbers of CD103 + dendritic cells and CX 3 CR1 + macrophages home to the gut mucosa. In contrast to innate expression of the antimicrobial lymphocytes, regulatory T (T Reg ) cells populate the intestinal mucosa in response to bacterial colonization. Although B cells are peptide REG3γ by epithelial present in gut tissue during early development, plasma cells producing dimeric IgA are only generated after birth to provide cells through secretion of IL 22. secretory IgA (SIgA) to the lumen. Maternal SIgA is provided by breast milk during the early postnatal period. NATURE REVIEWS IMMUNOLOGY VOLUME 12 JANUARY

4 Cryptopatches Clusters of KIT + IL 7R + THY1 + T cell progenitors found in the murine intestinal lamina propria. Cryptopatches are absent in germ-free mice. Isolated lymphoid follicles Small lymphoid aggregates located in the lamina propria of the small and large intestines that contain B cells, dendritic cells, stromal cells and some T cells and that might form germinal centres. In mice, isolated lymphoid follicles were shown to develop from cryptopatches and they are absent in germ-free animals. Anaphylaxis A severe and rapid allergic reaction triggered by the activation of high-affinity Fc receptors for IgE in sensitized individuals. Anaphylactic shock is the most severe type of anaphylaxis and can lead to death in minutes if left untreated. In various mouse models, it has been shown that IgG1 rather than IgE antibodies trigger the anaphylactic reaction. In parallel with bacterial colonization, the homing of lymphocytes to the gut mucosa seems to follow defined kinetics that indicate that a series of exogenous and endogenous signals regulates postnatal immune maturation in the intestine. Innate cells such as lymphoid tissue inducer (LTi) cells, natural killer (NK) cells, NK like NKp46 + cells and T helper 2 (T H 2)-like cells migrate during the first 4 weeks after birth from the murine fetal liver to the gut mucosa driven by endogenous signals 41. By contrast, the recruitment of forkhead box protein 3 (FOXP3) + regulatory T (T Reg ) cells to the gut mucosa and secretion of the anti-inflammatory cytokine IL 10 have been associated with bacterial colonization 42,43, whereby T Reg cells help to keep proinflammatory T H cells under control to preserve the epithelial barrier 44. A large body of evidence indicates that there are certain windows of opportunity for the induction of immunological tolerance in the gut and airways, particularly during the perinatal period (although reprogramming of the immune system might, in fact, be a life-long process). Oral tolerance has been best studied in mice and seems to be part of the normal immune maturation process, depending mainly on a finely tuned cross-talk between the innate and adaptive immune systems (in terms of antigen-presenting cells and T cells, respectively), as well as on epithelial barrier integrity 45. The role of T Reg cells. T Reg cells with immunosuppressive properties are abundant in human fetal MLNs 46, and their homing to the gut mucosa seems to be particularly active in infancy 47. Mechanistically, it is thought that CD103 + migratory dendritic cells (DCs) carry antigen from the gut to MLNs where they promote the induction of T Reg cells, particularly in the prescence of retinoic acid and TGFβ. In addition, subepithelial non-migratory CD103 CX 3 CR1 + macrophage-like cells produce IL 10, which supports proliferation of the T Reg cells when they have homed to the lamina propria 48. The increasing prevalence of allergic disorders in infancy indicates that the underlying immune dysregulation is probably an early event that compromises the function of immature antigen-presenting cells and T Reg cells 49,50. Bifidobacterium infantis, a prominent member of the gut microbiota in human infants, was shown in mice to markedly induce FOXP3 + T Reg cells 51. Notably, neonatal CD4 + T cells in mice are prone to differentiate into T Reg cells following stimulation 52, as are human cord blood cells, probably as a result of perinatal exposure to maternal progesterone 53. Later in development, members of the Clostridium cluster IV and XIVa might take over the role of B. infantis in promoting the local induction of T Reg cells in the colon 42. Also, Bacteroides fragilis seems to have unique T Reg cell-inducing and epitheliumassociating properties. Conversely, pro-inflammatory T H 17 cells are strongly promoted in the murine gut by segmented filamentous bacteria (SFB) 54,55. These bacteria, which attach to the mucosa particularly in the distal ileum, are mainly seen in mice after weaning 56. It is currently debated whether equivalents of mouse SFB are found in the human gut. The role of humoral immunity. Humoral immunity contributes to the establishment of an adequate postnatal epithelial barrier at mucosal surfaces. By reinforcing the epithelial barrier, secretory IgA (SIgA) inhibits inappropriate immune activation by microorganisms and antigens in the lumen of the intestinal and respiratory tracts. A large proportion of commensal microorganisms in the upper aerodigestive tract 57 and gut 58 is coated with SIgA 59. This bacterial coating might be explained by lowaffinity cross-reacting antibodies as well as by the high glycan content of SIgA 60. SIgA at the epithelial surface restricts colonization of microorganisms and the penetration of agents that could potentially cause hypersensitivity reactions or infection. Neonates that are not breast-fed lack reinforcement of the gut barrier by maternal SIgA 61. Plasma cells that produce IgA are generally undetectable in human lamina propria before 10 days of age and only traces of endogenously synthesized luminal SIgA and some SIgM are found after birth 45. The postnatal proliferation of intestinal IgA + plasma cells is highly variable, and in affluent societies it can take several years for the size of the IgA-producing plasma cell population to reach that of healthy adults 62. By contrast, a relatively fast postnatal increase in the production of SIgA can be seen in children living in developing countries with a heavy microbial load 63. Similarly, the number of intestinal IgA + plasma cells normalizes 4 weeks after colonization of germ-free mice with commensal microorganisms 64. Interestingly, pioneering studies in mice showed that the microbiota stimulates a self-limiting SIgA response in the gut 65. Such transient SIgA production is probably necessary to allow access of microbial constituents to gut-associated lymphoid tissue. In this manner, it seems that the intestinal IgA response is continuously adapting to the changing microbiota 66, which would be particularly important in the early postnatal period. Similar to the induction of T Reg cells, the establishment of SIgA-mediated immunity also depends on environmental conditions. For example, the differentiation of IgA + plasma cells depends on retinoic acid, so an optimal intestinal immune response requires adequate supply of vitamin A 67. The role of intestinal barrier reinforcement. The epithelial glycoprotein polymeric immunoglobulin receptor (pigr; also known as membrane secretory component) translocates SIgA and SIgM to the lumen and is therefore essential for the barrier function of the intestinal and respiratory mucosae 68. Mice deficient for pigr lack secretory-type antibodies 69 and have aberrant mucosal leakiness and excessive uptake of food proteins, as well as of components of commensal bacteria, from the gut lumen 69,70. This results in a hyper-reactive state and predisposition to anaphylaxis after systemic sensitization 71. Interestingly, pigr-deficient mice have increased induction of T Reg cells after continuous feeding of a soluble dietary antigen; this enhanced establishment of oral tolerance was shown to efficiently control IgG1- and T cell-dependent hypersensitivity JANUARY 2012 VOLUME 12

5 Tight junctions These are specialized intercellular junctions that seal the apical epithelium, in which two plasma membranes form a sealing gasket around a cell (also known as the zonula occludens). They are formed by several proteins, including occludin and claudin. Tight junctions prevent fluid moving through the intercellular gaps and prevent the lateral movement of membrane proteins between the apical and basolateral cellular domains. Goblet cells Mucus-producing cells that are found in the epithelial cell lining of the intestines and lungs. Clara cells Dome-shaped cells with short microvilli that are found in the small airways (bronchioles) of the lungs. These cells can secrete glycosaminoglycans to protect the lining of the bronchioles and are also known as bronchiolar exocrine cells. In adddition, decreased integrity of the structural barrier as a result of mild or transient breaching of the tight junctions between intestinal epithelial cells in mice was shown to induce an anti-inflammatory T Reg cell response in the gut 72. So, a predisposition to hypersensitivity might be compensated for by the enhanced induction of intestinal T Reg cells that promote tolerance to dietary antigens. These studies are relevant for newborn human infants, who have a leaky gut epithelium 45. Food allergy is much more prevalent in young children than in adults, but many children with food allergy outgrow their disorder before 3 years of age, particularly those with non-ige-mediated allergy to cow s milk 45. In these allergic children, and also in those who outgrow their IgE-mediated cow s milk allergy, the expansion of T Reg cell populations with suppressive properties has been observed 73,74. This indicates that a leaky neonatal gut epithelium and the concurrent microbial colonization might provide a window of opportunity for oral tolerance induction by continuous intestinal exposure to small amounts of dietary antigen. Of note, there are significant differences in the tightness of the epithelial barrier between mice and humans. In general, the gut mucosa shields the systemic immune system from exposure to constituents of commensal bacteria to a greater extent in mice than in humans 75. This is, however, not true for the immediate postnatal period. The so called gut closure the establishment of a mature epithelial barrier that prevents excessive influx of macromolecules across the intestinal epithelium develops during the first days after birth in humans but only with weaning in mice. Thus, some of the mechanisms that have been proposed for oral tolerance induction in mice, such as neonatal Fc receptor (FcRn)-mediated uptake of IgG antigen complexes from the gut lumen, are probably of little or no importance in humans. Dietary impact on perinatal gut immunity. Food constituents also contribute to the development of the intestinal immune effector compartments, both in mice and humans 76,77. Breast milk reinforces mucosal defences not only by providing SIgA (and SIgM) antibodies but also by delivering immune cells, cytokines, growth factors and high concentrations of oligosaccharides that promote the proliferation of lactic acidproducing bacteria, which are a major beneficial fraction of the neonatal intestinal microbiota 61. Indeed, a recent meta-analysis showed that breast-feeding has a protective effect on the development of inflammatory bowel disease later in life 78. Both breast milk and the maternal microbiome are transmitted to the neonate, but it is still unclear how the nutritional status of the mother affects the quality of her milk and the composition of her micobiota. It is known that intestinal microbial communities can be reshaped postnatally in response to changes in diet. Considerable efforts are therefore being made to examine the inter actions between food and food ingredients, the microbiota, the immune system and health 79. Vitamin A is a well-known dietary constituent that supports the mucosal milieu and facilitates the establishment of the neonatal immune system. The vitamin A derivative retinoic acid is required for the expression of gut-homing molecules on T and B cells (such as integrin α4β7 and CC-chemokine receptor 9 (CCR9)) 40, for the induction of T Reg cells and for IgA class switching in both mice and humans 40. Thus, an adequate supply of vitamin A is crucial for intestinal immune homeostasis. Taken together, the findings discussed above show that early postnatal events might significantly influence priming of the mucosal immune system and the establishment of a life-long immune homeostasis. Perinatal development of the airway mucosa The role of neonatal microbiota in airway immunity. Microbial colonization of the upper respiratory tract, including the nose, mouth and throat, occurs rapidly after birth. Until recently, the lower airways were thought to remain sterile, but recent findings have shown that microbial colonization also occurs in this compartment 9,10 (FIG. 2). As PRRs recognize microbial compounds and subsequently regulate immune responses to pathogens and commensals, it is thought that TLR expression is crucial for the functional development of the respiratory mucosa (FIG. 2). In the fetal mouse lung, TLR2 and TLR4 are first expressed during the last trimester of pregnancy (from the late pseudoglandular to terminal saccular stages), and expression levels increase further after birth 80. In humans, a comparable expression pattern was detected in the lung between early pseudoglandular and canalicular stages of fetal development (with the highest upregulation of TLR2 expression occurring between days 60 and 113 of gestation) 81. These TLRs are expressed by non-immune cells of the lung as well as by immune cells. Indeed, TLR4 expression by parenchymal lung cells is important for the normal development of lung elasticity and for airspace development, as shown in TLR4 deficient mice reconstituted with bone marrow from wild-type mice 82. What triggers prenatal TLR expression in the respiratory mucosa? It seems possible that microbial components, circulating in the maternal blood, cross the placental barrier to reach the amniotic fluid. Swallowing of amniotic fluid by the foetus could allow direct contact of such molecules with the developing respiratory surface. Although this is an attractive concept, so far there is no direct evidence in favour of this mechanism for the induction of prenatal TLR expression. Alternatively, an intrinsic developmental programme could be responsible for TLR expression. In either case, the presence of basal levels of TLR expression at birth is important to appropriately deal with environmental microorganisms that are inhaled with the first breath of life. The respiratory mucosal barrier. The airway epithelium is the first barrier layer to encounter immunostimulatory exogenous components at birth. Within the airway epithelium, ciliated columnar cells, mucus-secreting goblet cells and surfactant-secreting Clara cells are connected by tight junctions to form an impermeable but dynamic NATURE REVIEWS IMMUNOLOGY VOLUME 12 JANUARY

6 a Homeostasis c Birth Weaning Commensal bacterium Mucus Bacterial colonization Altered bacterial colonization IL-4 Epithelial cell DC IFNγ TGFβ IL-10 IL-17F TLR expression Altered TLR expression T H 2 cell T H 1 cell T Reg cell T H 17 cell Mucosal tolerance DC number Increased DC number b Chronic allergic inflammation of the respiratory mucosa Altered epithelial cell barrier function Aeroallergens TLR Altered density and diversity of commensal bacteria IL-4 Persistence of T H 2 cells ibalt formation IFNγ Delayed maturation of T H 1 cells Mast cell IgE-producing plasma cell TSLP IL-25 IL-33 ECP T H 2 cell Increased DC turnover and activation Eosinophil IL-5 IL-4 IL-13 IL-4 IL-5 IL-9 IL-13 Neutrophil IFNα IL-10 Prenatal airways Virus Virus Neonatal airways Virus Diminished antiviral response Virus Disturbed tolerance Adult airways Homeostasis Chronic allergic inflammation Figure 2 Development of the respiratory mucosal immune system. The establishment of airway homeostasis (a) or the development of chronic airway inflammation such as asthma (b) depends on early events that affect the maturation of the local immune system (c). Microbial colonization of the lower airways starts after birth (through inhalation). Maternal microbial exposure during pregnancy has an impact on prenatal immune programming (a and c). The expression of Toll-like receptors (TLRs) by airway epithelial cells starts during prenatal life and is further increased during postnatal life, influenced by exposure to microorganisms (c). TLR-triggered signalling cascades contribute to the development of the local dendritic cell (DC) network, which is not fully developed at birth (a and c). Exposure to high levels of TLR ligands favours the development of normal immune responses. The perinatal period is characterized by relatively high levels of expression of T helper 2 (T H 2) cell-associated cytokines, such as interleukin 4 (IL 4), which are rapidly down-regulated early in life under homeostatic conditions (c). T H 1 cell responses, as indicated by interferon-γ (IFNγ) secretion, develop following exposure to environmental factors, such as TLR agonists. In parallel, IL 10 secretion steadily increases after birth, leading to the development of mucosal tolerance (a and c). Lack of and/or altered prenatal maternal exposure to certain environmental microorganisms, Caesarean section and altered microbial colonization and diversity contribute to the development of asthma (b and c). These events result in altered TLR expression (c). Low-dose exposure to TLR agonists favours the development of T H 2 cell-mediated immune responses (b and c). T H 2 cell responses are further supported by the intrinsic adjuvant-like activities of certain aeroallergens, such as house dust mite proteins, leading to altered barrier function (b). TLR signalling cascades lead to the production of thymic stromal lymphopoietin (TSLP), IL 25 and IL 33 by airway epithelial cells, which support DC maturation and function (b). The chronic inflammatory immune response leads to the formation of induced bronchus-associated lymphoid tissue (ibalt), which participates in the initiation, maintenance and amplification of allergic inflammation in the respiratory mucosal tissue (b). This results in postnatal persistence of the prenatally developed T H 2 like immune responses. Moreover, T H 1 cell responses are markedly suppressed and/or delayed, and antiviral responses (IFNα) are diminished (c). ECP, eosinophil cationic protein; TGFβ, transforming growth factor-β, T Reg cell, regulatory T cell. 14 JANUARY 2012 VOLUME 12

7 barrier. Under inflammatory conditions, the permeability of the barrier is increased. The epithelial cells provide several nonspecific defence mechanisms, including the secretion of surfactants, complement products, antimicrobial peptides and mucins 2. It is now well recognized that the airway epithelium has an active role both in the initiation, maintenance and resolution of inflammatory mucosal responses, and in the repair of the respiratory epithelium, through the production of cytokines such as IL 25, IL 33 and thymic stromal lymphopoietin 83,84. In contrast to our more detailed knowledge of the expression of antimicrobial peptides in the gut early in life, we have only little information about their expression pattern in the airways. Young infants express β defensins and the cathelicidin LL37 and, although data are lacking regarding prenatal and early postnatal expression and function of these molecules, it is probable that there are no substantial differences between the mucosal systems of the gut and the airways in this regard 85. Perinatal maturation of the immune system. The organization of mucosa-associated lymphoid tissue has a distinct pattern in the bronchus compared with the gut. Classical bronchus-associated lymphoid tissue (BALT) as defined by B cell follicles and follicleassociated epithelium, with so-called membrane or microfold (M) cells 86,87 appears during late embryonic development in some species, but not in humans and mice 88,89. By contrast, the evidence indicates that de novo organization of lymphoid tissue is induced in murine and human lungs by the encounter of antigens in the airways during infections and inflammation 90,91. It is probable that DCs, at least in part, compensate for the lack of BALT in healthy human and mouse lungs in terms of immunological surveillance of the entire airway surface. Because the local DC network in the lungs develops with increasing amounts of antigen exposure, it is not surprising that DCs are absent or only present at low numbers before birth and continuously increase in number from birth onwards (FIG. 2). Therefore, at the time of birth in humans and mice, the respiratory mucosal barrier is only partly equipped to deal with environmental antigens, including bacteria, viruses and environmental allergens. Furthermore, indoor and outdoor air-pollutants, such as environmental tobacco smoke (ETS), have a marked effect on the development of perinatal immune functions (see below). As mentioned earlier, TLR expression is tightly regulated in the perinatal respiratory tract, and this affects the maturation of the adaptive mucosal immune system. A high-throughput study in humans of immune responses to a panel of TLR ligands in a cohort that was screened over the first 2 years of life showed that TLR ligation induces distinct T cell responses dependent on age 92. The levels of T H 1 type cytokines that are induced in response to TLR ligation are low at birth but increase gradually with age. The induction of interferon α (IFNα), which supports antiviral defences, reaches adult levels by 1 year of age. A similar progressive increase in the induction of IL 1β, tumour necrosis factor (TNF), IL 6 and IL 12 with age was also found 4,93. By contrast, IL 10 production steadily decreases from birth onwards, and the levels of IL 6 and IL 23 (which support the development of T H 17 cells) peak around the time of birth. These data indicate that the maturation of effector T cell responses occurs in an age-dependent manner under the control of mucosal TLR-mediated signalling (FIG. 2). This maturation process is particularly important to acquire the optimal level of T H 1 and T H 17 cells, which have a crucial role in defence against many pathogens. Furthermore, this response is required to suppress T H 2 cells, the levels of which are increased at birth. In conclusion, the regulation of TLR expression and function in the respiratory tract is closely associated with the development of innate and adaptive immune responses. Although microbial exposure clearly contributes to the development of normal mucosal immune responses, TLR expression in the respiratory mucosa occurs well before birth. Perinatal tolerance induction to allergens. It has long been known that exposure to aerosolized allergens (for example, ovalbumin) results in allergen-specific immune tolerance 94. Further studies have shown that this tolerance can be transferred from the mother to her offspring if antigen exposure occurs during pregnancy 95, but also if the mother has been tolerized before pregnancy 96. The effects of placental transfer of IgG antibodies have been extensively studied, and the results indicate that maternal antibodies can inhibit T cell responses in the offspring in an antigen-specific manner 95. Furthermore, placental transfer of allergens and antigens into the amniotic fluid, as well as the migration of maternal innate and adaptive immune cells, further facilitates immune development in the foetus 97. As a result of prenatal intra-uterine exposure to antigens, antigen-specific T cell responses of the neonate are readily detectable in cord blood. It is not yet clear whether this reflects tolerance development and/or affects the development of allergic sensitization and T H 2 type responses in the developing infant 98,99. Moreover, it has been shown in mice that the prenatal establishment of tolerance that protects from allergic asthma continues into the postnatal period through breast milk-mediated transfer of allergens 100. Also, alveolar epithelial cells can present inhaled antigens to T cells and promote the development of FOXP3 + T Reg cells 101 ; they can also produce IL 10, which induces the development of IL 10 producing T Reg cells. Thus, the development of tolerance against environmental allergens is a continuous process that starts in prenatal life. Tolerance-inducing mechanisms operating in the placenta during fetal life are supplemented from birth onwards by local immunoregulatory events that occur in the respiratory mucosa. Pathology of the gut mucosa Immunological hypersensitivity to food antigens. Several of the above-mentioned environmental factors influence the development of food allergies through modulating perinatal mucosal immunity. For example, birth by Caesarean section is associated with an altered intestinal microbiota in the offspring, who do NATURE REVIEWS IMMUNOLOGY VOLUME 12 JANUARY

8 not encounter maternal faecal vaginal commensal microorganisms during delivery 25,102,103. As a result, this form of delivery increases the risk of food allergy and coeliac disease 104,105 (BOX 1), probably because the resulting inadequate neonatal gut microbiota abrogates immune regulation. Interestingly, a small but significant decrease in the development of allergies was noted after perinatal probiotic intervention in children delivered by Caesarean section 106. Accordingly, it is possible that improved microbial intervention might be available in the future to rectify inadequate gut colonization, as observed after Caesarean section. However, other efforts to enhance oral tolerance by reinforcing the gut microbiota through probiotic treatment have had few convincing health benefits, even in children with a hereditary risk of food allergy. For example, no general inhibitory effect on the incidence of allergy was observed by the age of 5 years after perinatal probiotic intervention, which aimed to prevent T H 2 cell-driven IgE-mediated allergy 106. Thus, better molecular characterization of the tolerogenic components of the intestinal microbiota will be required for the advancement of microbial intervention as a preventive strategy. Interestingly, there might be a link between dietary lipids, such as the long-chain omega 3 fatty acids and short-chain fatty acids (SCFAs), and susceptibility to food allergies. Consumption of fish oil, which is enriched in polyunsaturated omega 3 fatty acids, might protect against the development of food allergies by decreasing immune cell recruitment and the production of pro-inflammatory cytokines in the gut 107. However, a positive correlation between maternal omega 3 fatty acid intake and the infant s protection from childhood asthma which often accompanies severe food allergy has not been shown in every study 108,109. In addition, SCFAs, which are produced by gut bacteria as a Box 1 Immune dysregulation in coeliac disease In certain parts of the world, coeliac disease is becoming more common, and both genetic and environmental factors are involved in the pathogensis. Central to the disease is T helper 1 (T H 1) cell-driven intestinal hypersensitivity to the immunodominant gliadin peptides in wheat gluten, or to similar prolamins in other types of cereal grain. The disease usually presents as malabsorption during childhood. The innate immune signals that cause the break in immune tolerance to prolamins and that initiate disease remain undefined 170. Without a prolamin-restricted diet, coeliac disease is a life-long disorder. In some patients, clinical symptoms do not occur until late in adulthood; this might reflect the possibility that initiating events, for example caused by repeated infections of the upper gastrointestinal tract, need to accumulate over time to break tolerance to prolamins. Extensive work has been carried out to identify other genetic susceptibility loci for coeliac disease, in addition to the well-defined HLA DQ2 (or less often HLA DQ8) MHC class II alleles, which mediate presentation of the proline-rich prolamin peptide epitopes to T H 1 cells. Several studies have linked hereditary single nucleotide polymorphisms (SNPs) in genes encoding various immune factors or innate immune sensors both with coeliac disease 171 and with other types of hypersensitivity to food or aeroallergens 172,173. This implies that aberrant innate immune responses are a crucial factor in the postnatal delay or abrogation of tolerance to food and other environmental allergens. Interestingly, some of the non-hla risk loci for coeliac disease are shared with autoimmune systemic disorders such as rheumatoid arthritis 171, which supports the idea that tolerogenic responses induced in the gut can disseminate beyond the gastrointestinal tract. byproduct of the fermentation of dietary fibres, have several health-promoting effects, in particular by strengthening the intestinal epithelial barrier 110. Indeed, food allergy in early childhood is associated with relatively low faecal levels of SCFAs, possibly as a result of delayed maturation of the intestinal microbiota 111. Lack of mucosal homeostasis in IBD. Numerous mouse models of IBD have identified a crucial role for both the gut microbiota and immune cells in its pathogenesis (FIG. 3). Despite the identification of disease-associated polymorphisms in genes that contribute to the intestinal epithelial barrier and immune homeostasis such as nucleotide-binding oligomerization domain 2 (NOD2), autophagy-related 16 like 1 (ATG16L1) and IL 23 receptor (IL23R) the clinical manifestations of IBD are usually only seen in late infancy or early adulthood. By contrast, patients with IL 10 or IL 10R deficiency suffer from early onset colitis 112. Thus, except in the case of total deficiency of IL 10 mediated signalling, the infant gut mucosa seems to have a greater capacity to control inappropriate immune responses than the adult mucosa, an ability that might be lost during repeated mucosal challenges later in life. Moreover, perinatal exogenous factors might contribute to the manifestation of overt disease symptoms later in life. These might be associated with postnatal environmental and microbial exposures, as children born by Caesarean section have recently been shown to have a slightly increased risk of developing Crohn s disease 113. Strikingly, certain alterations of the microbiota composition might be sufficient by themselves to cause mucosal inflammation, as the co-housing of animals that are genetically susceptible to colitis with neonatal or adult wild-type mice can lead to intestinal inflammation in the healthy immunocompetent animals 114,115. The colitogenic potential of an altered microbiota might be of particular importance in the neonatal and infant gut, where the immature microbiota is unstable and susceptible to colonization by additional bacteria. Another interesting observation is that mice carrying a mutation in the IBD-associated gene Atg16l1 only develop epithelial damage and enteric disease after infection with norovirus 116. A specific infection, potentially during the particularly susceptible postnatal period, might therefore alter the gut microbiota and/or lead to histological changes, both of which could trigger disease manifestation later in life in individuals with a genetic susceptibility. Thus, both infections and microbiota composition can contribute directly or indirectly to chronic inflammation 25,68,117. Necrotizing enterocolitis of the newborn. A striking example of the detrimental consequences of dysregulated perinatal maturation of the mucosal immune system is necrotizing enterocolitis (NEC) a disease that is observed in human pre-term neonates 118 and is characterized histologically by massive epithelial cell apoptosis. NEC has been associated with postnatal bacterial colonization. In animal models, altered epithelial TLR4 signalling has been linked to impaired stem cell proliferation 16 JANUARY 2012 VOLUME 12

9 a Intestinal mucosal homeostasis Intraepithelial lymphocyte b Chronic inflammation of the intestinal mucosa IAP PGRP2 CX 3 CR1 + macrophage CX3CR1 + macrophage SIgA Commensal bacterium REG3γ IL-10 TGFβ IL-10 TGFβ TSLP TGFβ RA T H 1 or T H 17 cell SIgA Tissue damage IgG IL-6 IL-1 IL-6 IL-12 IL-23 TSLP TGFβ T H 1 or T H 17 cell Mucus Defensins IL-22 TSLP TGFβ RA T Reg cell TGFβ RA Tolerogenic CD103 + DC Defensins REG3γ IgGproducing plasma cell IL-17A/F IFNγ T Reg cell TSLP TGFβ Paneth cell NKp46 + CD4 + T cell RORγt + lymphocyte IgA-producing plasma cell Neutrophils IgA-producing plasma cell BAFF APRIL RA Figure 3 The cellular network of gut immune homeostasis and the cellular processes driving mucosal inflammation. a Several mechanisms maintain microbial host homeostasis in mucosal tissues. The production of dimeric IgA by plasma cells followed by its epithelial transport into the gut lumen as secretory IgA (SIgA) is enhanced by retinoic acid (RA), B cell-activating factor (BAFF) and a proliferation-inducing ligand (APRIL); SIgA limits the epithelial translocation of antigen and bacteria. The mucus layer and constitutive α-defensin production shield the epithelial surface from direct microbial exposure. In addition, interleukin 22 (IL 22) secretion by NKp46 + RORγt + innate lymphocytes, CD4 + T cells and CD103 + dendritic cells (DCs) promotes epithelial production of regenerating islet-derived protein 3γ (REG3γ). Inappropriate immune activation is controlled by the enzymatic degradation of stimulatory microbial constituents by intestinal alkaline phosphatase (IAP) or the amidase peptidoglycan recognition protein 2 (PGRP2). Epithelial cell-derived regulatory factors, such as thymic stromal lymphopoietin (TSLP), transforming growth factor β (TGFβ) and retinoic acid, induce tolerogenic CD103 + DCs that in turn promote regulatory T (T Reg ) cell development. Both T Reg cells and CX 3 CR1 + macrophages control pro-inflammatory T helper 1 (T H 1) and T H 17 cells through the production of TGFβ and IL 10. b Microbial infection or tissue damage involving disruption of the epithelial barrier stimulates the release of antimicrobial peptides and abrogates the tolerogenic properties of CD103 + DCs by inducing their secretion of IL 6, IL 12 and IL 23. Together with IL 6 and IL 1 secretion by activated CX 3 CR1 + macrophages, this promotes the emergence of interferon-γ (IFNγ)- and IL 17-producing T H 1 and T H 17 cells, which in turn stimulate a pro-inflammatory reaction involving the infiltration of neutrophils and IgG-producing plasma cells, tissue destruction and, potentially, organ dysfunction. and crypt villus migration and decreased goblet cell differentiation 119. Interestingly, oral administration of Gram-positive probiotic bacteria has been shown to decrease the incidence of NEC 120. Although NEC does not lead to chronic inflammation later in life (after the affected gut tissue has been surgically removed), it illustrates the requirement of a mature, fully differentiated mucosal tissue for the establishment of mucosal homeostasis during postnatal microbial colonization. Less pronounced disturbances of mucosal maturation might induce similar processes and contribute to chronic inflammation later in life. NATURE REVIEWS IMMUNOLOGY VOLUME 12 JANUARY