The impact of perinatal immune development on mucosal ...manyan/To print/The impact of perinatal immune... · natal development in mice. During the first two weeks of postnatal life,

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 societies2,3. 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 contactmediated 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 importance7,8. Although current research focuses mainly on the intestinal microbiome, the functional relevance of the respiratory microbiome is also emerging9,10.

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 stimulation11–13.

1Institute of Laboratory Medicine and Pathobiochemistry, Molecular Diagnostics, Philipps University Marburg, Medical Faculty, Baldingerstrasse, 35043 Marburg, Germany.2Laboratory for Immunohistochemistry and Immunopathology (LIIPAT), Centre for Immune Regulation (CIR), University of Oslo, and Department of Pathology, Oslo University Hospital, Rikshospitalet, Oslo, Norway.3Institute for Medical Microbiology and Hospital Epidemiology, Hannover Medical School, Hannover, Germany.Correspondence to H.R. e-mail: [email protected]:10.1038/nri3112Published online 9 December 2011

The impact of perinatal immune development on mucosal homeostasis and chronic inflammationHarald Renz1, Per Brandtzaeg2 and Mathias Hornef3

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.

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Oral toleranceThis 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 architectureCrypts 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.

CathelicidinCathelicidins 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.

DefensinsA 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 mucosaThe 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 10–12 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 differentiation14,15. Also, the enteric spectrum of antimicrobial peptides changes significantly during neonatal development in mice. During the first two weeks of postnatal life, when mature cryptbased Paneth cells are absent16,17, the mouse intestinal epithelium expresses cathelicidinrelated 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 gut19, expression of another antimicrobial peptide by epithelial cells of the adult intestine — the Ctype lectin regenerating isletderived protein 3γ (REG3γ) — requires bacterial colonization of the intestine and is supported by interleukin22 (IL22)producing RORγt+NKp46+ lymphocytes20,21. 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 neonates22. 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 life23 (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 substrates24,25. 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 bacteria25. With weaning, an increasingly diverse microbiota is established that is highly individual and remains relatively stable throughout life26,27.

Perinatal microbiota–epithelial crosstalk. Exposure of mouse pups during or shortly after natural birth to lipopoly saccharide (LPS) from Gramnegative bacteria induces a transient transcriptional activation of the small intestinal epithelium with upregulated expression of microRNA146a (miR146a). Increased levels of miR146a in intestinal epithelial cells cause translational repression of the Tolllike receptor (TLR) signalling molecule IL1associated kinase 1 (IRAK1). This, together with the proteasomal degradation of IRAK1, contributes to innate immune tolerance by inhibiting TLR signalling28. 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 birth29 (FIG. 1). Conversely, epithelial expression of the nuclear factorκB inhibitor IκBα steadily increases during the postnatal period30. 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 miR146a expression and simultaneously induces sustained expression of genes supporting cell maturation, survival and nutrient absorption31. Innate immune signalling by epithelial cells seems to be essential for immune tolerance, as lack of the proinflammatory signalling molecule transforming growth factorβ (TGFβ)activated kinase 1 (TAK1) specifically in the murine intestinal epithelium leads to early inflammation, tissue damage and postnatal mortality32. 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 fortification33,34.

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 (RIGI) 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 gut37–39. Also, cryptopatches and isolated lymphoid follicles are seen only postnatally in mice40.

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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 the postnatal 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 intestinal expression level of the nuclear factor-κB inhibitor IκBα during the postnatal period decrease the responsiveness to bacterial lipopolysaccharide and other pro-inflammatory stimuli. Simultaneously, the acquisition of epithelial TLR tolerance creates a neonatal period of decreased innate immune responsiveness. Note that the human small intestinal epithelium at birth has a much more mature phenotype than in mice. The secondary lymphoid structures of Peyer’s patches and lymph nodes are generated before birth in mice and humans but mature during the postnatal period. By contrast, cryptopatches and isolated lymphoid follicles (ILFs) are formed after birth in mice. Specialized epithelial cells, known as M cells, reside above ILFs and Peyer’s patches and facilitate antigen transport from the lumen to the underlying lymphoid cells. Simultaneously, innate lymphocytes (such as lymphoid tissue inducer (LTi) cells) and T cells leave the liver and thymus, respectively, and colonize the enteric mucosal tissue, including the epithelium. Intraepithelial lymphocytes (IELs) reside in close proximity to the epithelium. Also, increasing numbers of CD103+ dendritic cells and CX

3CR1+ macrophages home to the gut mucosa. In contrast to innate

lymphocytes, regulatory T (TReg

) cells populate the intestinal mucosa in response to bacterial colonization. Although B cells are present in gut tissue during early development, plasma cells producing dimeric IgA are only generated after birth to provide secretory IgA (SIgA) to the lumen. Maternal SIgA is provided by breast milk during the early postnatal period.

RORγt+NKp46+ lymphocytesA population of innate lymphoid cells in the intestinal lamina propria that expresses the transcription factor retinoic acid receptor-related orphan receptor-γt (RORγt) and the natural killer cell marker NKp46. RORγt+NKp46+ lymphocytes support the expression of the antimicrobial peptide REG3γ by epithelial cells through secretion of IL-22.

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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 folliclesSmall 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.

AnaphylaxisA 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, NKlike NKp46+ cells and T helper 2 (TH2)like cells — migrate during the first 4 weeks after birth from the murine fetal liver to the gut mucosa driven by endogenous signals41. By contrast, the recruitment of forkhead box protein 3 (FOXP3)+ regulatory T (TReg) cells to the gut mucosa and secretion of the antiinflammatory cytokine IL10 have been associated with bacterial colonization42,43, whereby TReg cells help to keep proinflammatory TH cells under control to preserve the epithelial barrier44.

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 lifelong 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 crosstalk between the innate and adaptive immune systems (in terms of antigenpresenting cells and T cells, respectively), as well as on epithelial barrier integrity45.

The role of TReg cells. TReg cells with immunosuppressive properties are abundant in human fetal MLNs46, and their homing to the gut mucosa seems to be particularly active in infancy47. Mechanistically, it is thought that CD103+ migratory dendritic cells (DCs) carry antigen from the gut to MLNs where they promote the induction of TReg cells, particularly in the prescence of retinoic acid and TGFβ. In addition, subepithelial nonmigratory CD103–CX3CR1+ macrophagelike cells produce IL10, which supports proliferation of the TReg cells when they have homed to the lamina propria48. 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 antigenpresenting cells and TReg cells49,50.

Bifidobacterium infantis, a prominent member of the gut microbiota in human infants, was shown in mice to markedly induce FOXP3+ TReg cells51. Notably, neonatal CD4+ T cells in mice are prone to differentiate into TReg cells following stimulation52, as are human cord blood cells, probably as a result of perinatal exposure to maternal progesterone53. 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 TReg cells in the colon42. Also, Bacteroides fragilis seems to have unique TReg cellinducing and epithelium associating properties. Conversely, proinflammatory TH17 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 weaning56. 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 tract57 and gut58 is coated with SIgA59. This bacterial coating might be explained by lowaffinity crossreacting antibodies as well as by the high glycan content of SIgA60. 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 breastfed lack reinforcement of the gut barrier by maternal SIgA61. 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 birth45. 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 IgAproducing plasma cell population to reach that of healthy adults62. 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 load63. Similarly, the number of intestinal IgA+ plasma cells normalizes 4 weeks after colonization of germfree mice with commensal microorganisms64. Interestingly, pioneering studies in mice showed that the microbiota stimulates a selflimiting SIgA response in the gut65. Such transient SIgA production is probably necessary to allow access of microbial constituents to gutassociated lymphoid tissue. In this manner, it seems that the intestinal IgA response is continuously adapting to the changing microbiota66, which would be particularly important in the early postnatal period.

Similar to the induction of TReg cells, the establishment of SIgAmediated 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 A67.

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 mucosae68. Mice deficient for pIgR lack secretorytype antibodies69 and have aberrant mucosal ‘leakiness’ and excessive uptake of food proteins, as well as of components of commensal bacteria, from the gut lumen69,70. This results in a hyperreactive state and predisposition to anaphylaxis after systemic sensitization71. Interestingly, pIgRdeficient mice have increased induction of TReg cells after continuous feeding of a soluble dietary antigen; this enhanced establishment of oral tolerance was shown to efficiently control IgG1 and T celldependent hypersensitivity71.

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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 cellsDome-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 antiinflammatory TReg cell response in the gut72. So, a predisposition to hypersensitivity might be compensated for by the enhanced induction of intestinal TReg cells that promote tolerance to dietary antigens.

These studies are relevant for newborn human infants, who have a leaky gut epithelium45. 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 nonIgEmediated allergy to cow’s milk45. In these allergic children, and also in those who outgrow their IgEmediated cow’s milk allergy, the expansion of TReg cell populations with suppressive properties has been observed73,74. 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 humans75. 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 humans76,77. 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 acid producing bacteria, which are a major beneficial fraction of the neonatal intestinal microbiota61. Indeed, a recent metaanalysis showed that breastfeeding has a protective effect on the development of inflammatory bowel disease later in life78.

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 health79.

Vitamin A is a wellknown 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 guthoming molecules on T and B cells (such as integrin α4β7 and CCchemokine receptor 9 (CCR9))40, for the induction of TReg cells and for IgA class switching in both mice and humans40. 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 lifelong immune homeostasis.

Perinatal development of the airway mucosaThe 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 compartment9,10 (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 birth80. 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 nonimmune 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 TLR4deficient mice reconstituted with bone marrow from wildtype mice82.

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, mucussecreting goblet cells and surfactantsecreting Clara cells are connected by tight junctions to form an impermeable but dynamic

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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

H2) cell-associated

cytokines, such as interleukin-4 (IL-4), which are rapidly down-regulated early in life under homeostatic conditions (c). T

H1 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

H2

cell-mediated immune responses (b and c). TH2 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

H2-like

immune responses. Moreover, TH1 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.

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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 mucins2. 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 IL25, IL33 and thymic stromal lymphopoietin83,84. 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 βdefensins 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 regard85.

Perinatal maturation of the immune system. The organization of mucosaassociated lymphoid tissue has a distinct pattern in the bronchus compared with the gut. Classical bronchusassociated lymphoid tissue (BALT) — as defined by B cell follicles and follicleassociated epithelium, with socalled membrane or microfold (M) cells86,87 — appears during late embryonic development in some species, but not in humans and mice88,89. 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 inflammation90,91. 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 airpollutants, 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 highthroughput 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 age92. The levels of TH1type cytokines that are induced in response to TLR ligation are low at birth but increase gradually with age. The induction of interferonα (IFNα), which supports antiviral defences, reaches adult levels by 1 year of age. A similar progressive increase in the induction of IL1β, tumour necrosis factor (TNF), IL6 and IL12 with age was also found4,93. By contrast, IL10

production steadily decreases from birth onwards, and the levels of IL6 and IL23 (which support the development of TH17 cells) peak around the time of birth. These data indicate that the maturation of effector T cell responses occurs in an agedependent manner under the control of mucosal TLRmediated signalling (FIG. 2). This maturation process is particularly important to acquire the optimal level of TH1 and TH17 cells, which have a crucial role in defence against many pathogens. Furthermore, this response is required to suppress TH2 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 allergenspecific immune tolerance94. Further studies have shown that this tolerance can be transferred from the mother to her offspring if antigen exposure occurs during pregnancy95, but also if the mother has been tolerized before pregnancy96. 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 antigenspecific manner95. 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 foetus97. As a result of prenatal intrauterine exposure to antigens, antigenspecific 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 TH2type responses in the developing infant98,99.

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 milkmediated transfer of allergens100. Also, alveolar epithelial cells can present inhaled antigens to T cells and promote the development of FOXP3+ TReg cells101; they can also produce IL10, which induces the development of IL10producing TReg cells. Thus, the development of tolerance against environmental allergens is a continuous process that starts in prenatal life. Toleranceinducing 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 mucosaImmunological hypersensitivity to food antigens. Several of the abovementioned 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

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not encounter maternal faecal–vaginal commensal microorganisms during delivery25,102,103. As a result, this form of delivery increases the risk of food allergy and coeliac disease104,105 (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 section106. 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 TH2 celldriven IgEmediated allergy106. 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 longchain omega3 fatty acids and shortchain fatty acids (SCFAs), and susceptibility to food allergies. Consumption of fish oil, which is enriched in polyunsaturated omega3 fatty acids, might protect against the development of food allergies by decreasing immune cell recruitment and the production of proinflammatory cytokines in the gut107. However, a positive correlation between maternal omega3 fatty acid intake and the infant’s protection from childhood asthma — which often accompanies severe food allergy — has not been shown in every study108,109. In addition, SCFAs, which are produced by gut bacteria as a

byproduct of the fermentation of dietary fibres, have several healthpromoting effects, in particular by strengthening the intestinal epithelial barrier110. 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 microbiota111.

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 diseaseassociated polymorphisms in genes that contribute to the intestinal epithelial barrier and immune homeostasis — such as nucleotidebinding oligomerization domain 2 (NOD2), autophagyrelated 16like 1 (ATG16L1) and IL23 receptor (IL23R) — the clinical manifestations of IBD are usually only seen in late infancy or early adulthood. By contrast, patients with IL10 or IL10R deficiency suffer from early onset colitis112. Thus, except in the case of total deficiency of IL10mediated 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 disease113.

Strikingly, certain alterations of the microbiota composition might be sufficient by themselves to cause mucosal inflammation, as the cohousing of animals that are genetically susceptible to colitis with neonatal or adult wildtype mice can lead to intestinal inflammation in the healthy immunocompetent animals114,115. 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 IBDassociated gene Atg16l1 only develop epithelial damage and enteric disease after infection with norovirus116. 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 inflammation25,68,117.

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 preterm neonates118 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

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

H1) 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 undefined170. 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

H1 cells. Several studies have linked hereditary single nucleotide

polymorphisms (SNPs) in genes encoding various immune factors or innate immune sensors both with coeliac disease171 and with other types of hypersensitivity to food or aeroallergens172,173. 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 arthritis171, which supports the idea that tolerogenic responses induced in the gut can disseminate beyond the gastrointestinal tract.

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TReg cellTReg cell

IL-10TGFβ

IL-10TGFβ

TSLPTGFβRA

TSLPTGFβ

IL-6IL-1

IL-6IL-12IL-23

TSLPTGFβRA

TSLPTGFβ

Tolerogenic CD103+ DC

CX3CR1+

macrophageCX3CR1+

macrophage

IgA-producing plasma cell

IgA-producing plasma cell

Neutrophils

REG3γ

REG3γ

Defensins

IL-17A/FIFNγ

NKp46+

RORγt+

lymphocyte

Paneth cell

BAFFAPRILRA

IL-22

TGFβRA

a Intestinal mucosal homeostasis

Commensalbacterium

IAPPGRP2

Intraepitheliallymphocyte

CD4+ T cell

SIgA

Mucus

SIgA

IgG TH1 orTH17 cell

Tissuedamage

Defensins

TH1 orTH17 cell

b Chronic inflammation of the intestinal mucosa

IgG-producing plasma cell

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

TReg

cells and CX3CR1+ macrophages control pro-inflammatory T helper 1 (T

H1) and T

H17 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

3CR1+ macrophages, this promotes the emergence of

interferon-γ (IFNγ)- and IL-17-producing TH1 and T

H17 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 differentiation119. Interestingly, oral administration of Grampositive probiotic bacteria has been shown to decrease the incidence of NEC120. 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.

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Pathology of the respiratory mucosaThe role of commensals in allergy and asthma develop-ment. Neonatal intestinal colonization with an altered microbiota can influence susceptibility to allergy and asthma, and the mechanisms involved are currently under investigation (TABLE 1). Differences in the composition of the gut microbiota between atopic or allergic and nonatopic or nonallergic infants have been described in several models; for example, lower counts of Lactobacillae, Bifidobacteriae and Bacteroides species, together with higher counts of Clostridium dif-ficile, were found in atopic infants121–125. In keeping with this influence of the gut microbiota, the epidemiological association between the disappearance of Helicobacter pylori from the human stomach and the increasing incidence of allergic asthma in Western societies has recently been verified as causal in an allergen induced mouse model126. Of note, the accumulation of TReg cells in the lungs and protection against allergic asthma was most robust in mice infected neonatally with H. pylori. It is important to note that most of these studies used classical culturebased techniques to monitor microbiota composition. It is expected that the use of nonculturebased techniques to qualitatively and quantitatively assess the gut microbiome will shed new light on the relationship between the gut microbiota and allergy.

The effect of intestinal commensals on asthma development has been elegantly illustrated in mice: depletion of the gut microbiota by antibiotic treatment exacerbated experimental asthma that is driven by the TH2 cellassociated cytokine IL13 (REF. 127). Moreover, a recent study provided a molecular link between diet, gastrointestinal bacterial metabolism and asthma development128. SCFAs, which are produced through the fermentation of dietary fibres by the intestinal microbiota (namely, Bifidobacteriae and Bacteroides species), bind to Gproteincoupled receptor 43 (GPR43); in the absence of this interaction — as in Gpr43–/– mice — exacerbation of experimental asthma was observed. This was related to an increased production of proinflammatory mediators and increased immune cell recruitment in the airways.

Finally, the (uro)genital microbiome of the mother might be involved in protection against asthma in her offspring. This hypothesis is receiving increased attention owing to the observation of a higher risk of asthma in children delivered by Caesarean section129–131.

Environmental microbial exposure. In addition to the microorganisms that colonize the mucosal surfaces, the inhalation of airborne environmental microorganisms can also trigger mucosal immune maturation (FIG. 2). Earlylife exposure to the unique environment of traditional farming was found to significantly decrease the risk of developing respiratory allergies (reviewed in REF. 132). The high microbial load that is present in the farm environment is strongly associated with increased production of IFNγ and TNF at birth, but was found to have no effect on the levels of TH2type cytokines or on the production of IL10 and IL12. The data regarding IgE production by these children after birth or during early childhood are inconsistent133,134; however, an inverse relationship between neonatal IgE production and the level of cord blood IFNγ was noted135, which further supports the concept that early development of a strong TH1type immune response is an important mechanism to suppress the initial polarization of T cells towards a TH2type response and to further inhibit the development of TH2 cellmediated allergies.

Exposure to environmental microorganisms during early life has a large effect on the level of TLR expression, as indicated by increased levels of Tlr5 and Tlr9 mRNA at birth in exposed compared with nonexposed newborn mice (FIG. 2). When microbial exposure continues through early childhood, the effects are even greater, as shown in the case of natural exposure to endotoxin136 derived from mattress dust. Moreover, the higher the number of different farm animal species that a mother had contact with during pregnancy, the higher are the levels of TLR2, TLR4 and CD14 expression in her offspring at school age137.

Furthermore, it has been shown that mucosal administration of natural or synthetic TLR agonists — such as lipopeptides (which stimulate TLR2)138, peptidoglycan (TLR2 and TLR4)139, LPS (TLR4)140,141, polyinosinic–polycytidylic acid R848 (TLR3 and TLR7)142 and DNA

Table 1 | Microorganisms affect the development of experimental and clinical asthma

Source and route of microbial exposure

Examples of bacteria Effect on asthma

Environmental microorganisms

• Acinetobacter lwoffii• Eurotium spp.• Penicillium spp.

• Transgenerational protection from experimental asthma• Asthma-protected children are exposed to a high level of

bacteria and fungi/spores103,146

Respiratory microbiota • Proteobacteria• Bacteroides spp.

• Altered microbial colonization pattern of the respiratory mucosa in asthmatics and patients with COPD9

Intestinal microbiota • Lactobacillae• Bifidobacteriae• Bacteroides spp.• Clostridium spp.

• Altered quantitative distribution pattern of certain culturable microbial strains in young asthmatic patients121–125

• Depletion of gut microbiota exacerbates experimental asthma127

Urogenital microbiota • Vaginal microorganisms • Indirect evidence from a model of Caesarean section; cause–effect relationship needs to be established129–131

COPD, chronic obstructive pulmonary disease.

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from Bordetella pertussis (TLR9)143 — triggers a cascade of immune responses that results in protection from experimental asthma in several murine models. In most of these studies, the absence of a TH2 cellmediated immune response correlated with the development of a strong TH1type immune response or increased levels of IL12 and IL10, further supporting the concept that TLR signalling favours the development of a TH1 cellmediated immune response (FIG. 2). Further studies in mice have provided evidence that a similar asthmaprotective effect could be achieved by intranasal application of intact Gramnegative or Grampositive bacteria144.

When infants are exposed to TLR ligands, their response pattern at the level of adaptive immunity differs markedly between healthy and allergic infants10,15; for example, allergic children who were exposed to TLR ligands at a young age had decreased production of proinflammatory cytokines (such as IL1β, TNF and IL6)145. Moreover, they had a markedly delayed maturation of TH1 cell functions (FIG. 2), which is, at least in part, responsible for the sustained and high TH2 cell response profile of allergic individuals (for example, owing to lack of IFNγ production).

These proofofprinciple experiments carried out in adult animals provide the foundation for the exploration of transmaternal asthmaprotective effects. Several asthmaprotective bacterial strains have been identified, but modulation of the asthmatic phenotype in mice showed that there are straindependent qualitative and quantitative differences, with the highest degree of protection so far observed by Acinetobacter lwoffii146. Intranasal administration of A. lwoffii to mice throughout pregnancy results in a strong and longlasting protection from airway inflammation, airway hyperresponsiveness and IgE production in their offspring. Moreover, it augmented TLR expression in the maternal lung, resulting in mild local and systemic inflammation, as marked by increased expression of IL6, TNF and IL12p40. This maternal mucosal TLR signalling was responsible for the initiation and eventual transmission of the asthmaprotective effect to the next generation, as TLRsufficient offspring of mothers who are deficient for TLR2, TLR3, TLR4, TLR7 and TLR9 were not protected from asthma146. Thus, TLR expression and signalling in the maternal airway mucosa are crucially involved in generating the asthmaprotective immune responses that operate in the offspring. Although the downstream events could be linked to a lowlevel inflammatory immune response, the detailed molecular and cellular events involved remain to be elucidated. Future studies must be carried out to determine the contribution of individual bacterial strains as opposed to the overall microbial burden and diversity in this regard103.

Perinatal tobacco smoke exposure. Environmental tobacco smoke is a major component of indoor air pollution. There is a strong association between maternal smoking during pregnancy, or postnatal ETS exposure, and a higher risk of asthma147,148. Studies in mice and monkeys show that intrauterine ETS exposure results in decreased lung development and an accelerated decline in lung function149 — with increased thickness of the smooth muscle layer and

collagen type III deposition around the airways — of the offspring150,151. Moreover, the expression of genes involved in WNT signalling was induced in the offspring of mice exposed to cigarette smoke during pregnancy152. These data imply that signalling pathways involved in inflammation and lung cancer development contribute to the detrimental effects of prenatal ETS exposure.

The development of the mucosal immune system in neonates is strongly affected by ETS exposure during pregnancy. Cells from neonates born to mothers who smoke have an impaired response to TLR ligands, resulting in the decreased production of proinflammatory cytokines by antigenpresenting cells153. This involves decreased production of TNF in response to TLR2, TLR3 and TLR4 signalling, decreased IL6 production in response to TLR2 and TLR9 stimulation, and decreased IL10 levels after TLR2 activation. These results are in line with previous reports showing that altered neonatal cytokine responses are associated with maternal smoking in pregnancy154, and the decreased production of IL10 by DCs of babies exposed to ETS in the first five months of life155. Moreover, there are some indications that levels of IFNγ might be decreased in the offspring of mothers who smoke during pregnancy153. The absence of IFNγ production might contribute to the higher levels of TH2type cytokines observed following antigen challenge in neonates154. Indeed, prenatal ETS exposure exacerbates experimental lung inflammation in mice through the induction of a strong TH2 cell response in the lung marked by increased levels of IL4, IL5, IL6 and IL13 and decreased levels of IL2 and IFNγ156 (FIG. 2).

In BALB/c mice, ETSexposed female neonates are significantly more susceptible than ETSexposed male neonates to allergic responses157 and to ETSdriven production of TH2type cytokines. This gender difference has been attributed to the TH2 cellenhancing activity of progesterone158. In addition, ETS exposure during the early postnatal period has strong immunosuppressive effects, particularly on T cells, resulting in impaired T cell receptor signalling159,160. It is probable that this effect dampens the normal development of T cell responses, particularly against environmental stimuli and microorganisms, as indicated by the increased susceptibility of smokers to respiratory infections and asthma exacerbation.

Furthermore, the predisposition to asthma can be increased by intrauterine ETS exposure combined with some genetic factors (such as polymorphisms in the genes encoding glutathioneStransferase160, in the functional promoter of TGFB1 (REF. 161) or in IL1R162). These are important examples of gene–environment interactions that result in the development of a clinical phenotype.

In conclusion, environmental factors have a marked effect on the development of perinatal mucosal immune responses in the respiratory tract. Prenatal exposure to dietary components might influence the pathogenesis of respiratory disease (BOX 2). Moreover, perinatal ETS exposure is one of the most important risk factors for asthma development in early life. In addition, perinatal exposure to commensal bacteria of the gut, airways, skin or vagina and airborne environmental microorganisms (FIG. 2) is the most prominent exogenous regulator of perinatal respiratory immunity. Other microorganisms, such as viruses

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and fungi, might also have an important role in mucosal immune responses but their contribution has not yet been extensively studied. Therefore, further research is needed to fully understand the impact of host–microbial interactions at the local mucosal surface on shaping innate and adaptive mucosal immune responses.

Emerging interventional approachesRecent research on epithelial homeostasis, mucosal immunity and the mucosal microbiota has revealed the enormous complexity of the host–microbial and host–environmental relationships. Better understanding of this complexity has paved the way for novel therapeutic or preventive measures. Antigenspecific approaches to reestablish tolerance in allergic patients through administration of the allergen as a ‘tolerogen’ have provided promising results in clinical studies45. Also, antigen nonspecific approaches, such as helminthderived immuno modulators, probiotics and diet regimen, might be important in the future treatment of immune mediated pathologies3,163, although the data are not yet conclusive. Experiments have indicated that some bacterial strains can induce DCs, directly or through

epithelial mediators, to exert antiinflammatory effects. In addition, early intestinal colonization with beneficial probiotic bacteria was shown to enhance SIgA export to the gut lumen164. Prebiotics, such as selected oligosaccharides, increase the concentration of beneficial bacteria and the levels of SIgA in healthy infants. However, the selection of the right probiotic bacterial strains, or of symbiotic combinations of prebiotics and breast milk, remains a difficult task and requires the consideration of several safety issues, as certain bacterial strains might be proinflammatory165,166. A more promising strategy might be to identify the underlying molecular mechanisms of immune tolerance and to administer the specific components or metabolites that enhance perinatal mucosal immune homeostasis167,168. Finally, another potential future approach for the treatment of chronic inflammatory disease is the interference with specific signal transduction pathways or transcription factors to reduce or promote a certain type of immune response169. Nevertheless, we are only just beginning to identify the cellular and molecular mechanisms that facilitate the postnatal establishment of homeostasis and, thus, potential targets for rational manipulation.

Box 2 | The role of dietary and nutritional components in the pathogenesis of allergy and asthma

There is increasing evidence that the pathogenesis of respiratory diseases, including asthma, might be associated with prenatal exposure to dietary components. The effects of vitamins A and D on asthma are being investigated.

Vitamin A has an essential role in cellular and subcellular membrane stability and, thus, influences the repair and development of epithelial cells174. However, it might promote an allergic response, as it has been shown to favour the development of T helper 2 (T

H2) cells175.

Vitamin D is important for lung maturation and surfactant production, which is of particular importance at the end of the intra-uterine phase of lung development, when the alveolar epithelium undergoes abrupt differentiation as part of the preparation for gas exchange at birth176,177. Furthermore, vitamin D and its metabolites have a broad range of immunomodulatory effects on both innate and adaptive immune cells178. Vitamin D induces tolerogenic dendritic cells that are characterized by the upregulation of expression of the inhibitory receptors immunoglobulin-like transcript 3 (ILT3) and ILT4 (REFS 179,180). A large cohort study181 associated vitamin D supplementation during pregnancy with a significant increase in the mRNA levels of ILT3 and ILT4 in cord blood.

However, the clinical implications of vitamin A and vitamin D supplementation during pregnancy still remain controversial182,183. Conflicting results might be related to the differential effects of the isoforms and metabolites of these vitamins. Clearly, more mechanistic studies are needed to explore the precise effects on fetal immune development.

A methyl donor-rich diet, consisting of high amounts of vitamin B12, folic acid, choline and methionine, might also influence the development of perinatal immunity184. Intra-uterine exposure to a high dose of methyl donors increased the rate of methylation at CpG DNA, which is an important mechanism of epigenetic regulation185. Supplementation of pregnant mice with such a methyl donor-rich diet increased the development of experimental asthma in the offspring186. However, the implications of these results for the development of human asthma remain a matter of debate187,188.

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AcknowledgementsH.R. is supported by the SFB/TR22 DFG-funded network ‘Allergic Immune Responses of the Lung’ and by the Universities of Giessen and Marburg Lung Centre (UGMLC), a LOEWE excellence initiative of the Hessian Government, Germany. P.B. is supported by the Research Council of Norway, The University of Oslo and Oslo University Hospital, through the joint financing of the Center of Excellence named ‘Centre for Immune Regulation’. M.H. is supported by the German Research Foundation (Ho2236/5-3), the Federal Ministry of Education and Research (DLR 01GU0825 and DLR 01KI1003D) and the Collaborative Research Center grants SFB621 and SFB900.

Competing interests statement The authors declare no competing financial interests.

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