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Innate immunity of the newborn: basic mechanisms and clinical correlates


Ofer Levy

Abstract | The fetus and newborn face a complex set of immunological demands, including protection against infection, avoidance of harmful inflammatory immune responses that can lead to pre-term delivery, and balancing the transition from a sterile intra-uterine environment to a world that is rich in foreign antigens. These demands shape a distinct neonatal innate immune system that is biased against the production of pro-inflammatory cytokines. This bias renders newborns at risk of infection and impairs responses to many vaccines. This Review describes innate immunity in newborns and discusses how this knowledge might be used to prevent and treat infection in this vulnerable population.
All organ systems of the body undergo a dramatic transition at birth, from a sheltered intra-uterine existence to the radically distinct environment of the outside world. This acute transition is then followed by a gradual, age-dependent maturation. The fetal and neonatal immune systems are associated with physiological demands that are three-fold: protection against infection, including viral and bacterial pathogens at the maternalfetal interface1,2, avoidance of potentially harmful pro-inflammatory/T helper 1 (T H 1)-cellpolarizing responses that can induce alloimmune reactions between mother and fetus3, and mediation of the transition between the normally sterile intra-uterine environment to the foreign antigen-rich environment of the outside world, including primary colonization of the skin4 and intestinal tract5 by microorganisms. Given the limited exposure to antigens in utero and the well-described defects in neonatal adaptive immunity 6, newborns must rely on their innate immune systems for protection to a significant extent7,8. As the innate immune system can instruct the adaptive immune response 9, distinct functional expression of neonatal innate immunity, including a bias against TH1-cell-polarizing cytokines, contributes to a distinct pattern of neonatal adaptive immune responses. Mounting evidence indicates that infection-induced production of pro-inflammatory/TH1-cell-polarizing cytokines, including tumour-necrosis factor (TNF) and interleukin-1 (IL-1 ) , is associated with premature labour and pre-term delivery10. In particular, TNF production is thought to favour abortion through the induction of apoptosis in placental and fetal cells. The ability of pro-inflammatory cytokines to induce spontaneous abortion is likely to be an important reason for the strong bias of the maternal and fetal immune systems of multiple mammalian species towards TH2-cell-polarizing cytokines8,11. Because of this impaired production of TH1-cellassociated cytokines, it was initially thought that the neonatal innate immune system was generally impaired or depressed; however, stimulus-induced production of certain cytokines (for example, IL-6, IL-10 and IL-23) by neonatal monocytes and antigenpresenting cells (APCs) actually exceeds that of adults1214. However, the bias against TH1-cell-polarizing cytokines leaves the newborn susceptible to microbial infection and contributes to the impairment of neonatal immune responses to most vaccines, thereby frustrating efforts to protect this vulnerable population 15. After birth, there is an age-dependent maturation of the immune response. Of note, prenatal and postnatal exposure to environmental microbial products that can activate innate immunity might accelerate this maturation process, particularly if the exposure occurs repeatedly over time16, diminishing TH2-cell polarization and/or enhancing TH1-cell polarization and thereby potentially reducing allergy and atopy, in accord with the hygiene hypothesis17,18. The uterine and placental tissues (BOX 1; FIG. 1) , fetus and newborn have a unique anatomical distribution and functional expression of innate immune molecules, including Toll-like receptors (TLRs; receptors that serve to detect the presence of microbes1921), cationic membrane-active antimicrobial proteins and peptides (APPs) with microbicidal and microbial toxin-neutralizing activities 22,23, and chemokines.

Hygiene hypothesis
The theory that exposure to microbial components, including Toll-like receptor (TLR) agonists during the neonatal, infancy and early-childhood phases of development serves to polarize the immune response towards T helper 1 (TH1)-cell, and away from TH2-cell, responses, thereby reducing the likelihood of allergy and/or atopy. Consistent with this hypothesis, there are inverse epidemiological relationships between the rates of infection and autoimmunity for example, as the rates of common infections have dropped in wealthy industrialized countries, the rates of allergy and autoimmune disease have risen.

Department of Medicine, Division of Infectious Diseases, Childrens Hospital Boston and Harvard Medical School, Boston, MA 02115, USA. e-mail: ofer.levy@childrens. harvard.edu doi:10.1038/nri2075

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Box 1 | Innate immunity of the uterus, placenta and amniotic fluid
The uterine cavity contains innate immune detection and effector systems that maintain sterility, detect infection and, under conditions of substantial microbial invasion, induce the expression of mediators that speed lung maturation and labour in order to deliver the fetus from a threatening environment (FIG. 1). The cervical plug, a post-conception barrier between the non-sterile vagina and the normally sterile intrauterine cavity, contains antimicrobial proteins and peptides (APPs; including lactoferrin, lysozyme and -defensins), which are thought to protect against ascending microbial infection139. Human uterine epithelial cells express Toll-like receptor 1 (TLR1) to TLR9, which are capable of mediating the robust production of interleukin-6 (IL-6) and IL-8 (cytokines that have important roles in cervical relaxation and labour140,141), as well as the production of -defensins and interferon-141. The trophoblast, a layer of fetal cells in the uterus that mediates the implantation of the fetus into the placenta, expresses TLR2 and TLR4. TLR2 activation on these cells induces apoptosis, indicating a pathway by which intra-uterine infections might contribute to complications of pregnancy such as intra-uterine growth restriction142,143. By contrast, activation of TLR4, which is expressed at the maternal-facing plasma membrane that is bathed in maternal blood144, results in cytokine production142 and might mediate responses to maternal infection. The amniotic fluid contains acutephase proteins, such as soluble CD14 and lipopolysaccharide (LPS)-binding protein (LBP)145 that modulate the endotoxic activity of LPS81, as well as APPs, such as lactoferrin, bactericidal/permeability-increasing protein (BPI), histones146 and defensins147,148. Pre-term labour increases amniotic-fluid concentrations of the 14 kDa group II phospholipase A2 (PLA2)149, an enzyme with remarkable potency against Gram-positive bacteria150. Consistent with its content of multiple APPs, amniotic fluid possesses antimicrobial activity in vitro against multiple Gram-negative and Gram-positive bacterial species151.

Such innate immune molecules are increasingly implicated in human health and disease and are currently the subject of intense biopharmaceutical research23,24. Therefore, the study of neonatal innate immunity is of interest, not only as a biological phenomenon, but also in identifying new methods to diagnose, prevent and treat infection and allergy in the newborn and infant. Here we focus on the distinct anatomical expression of molecules associated with newborn innate immunity and on unique aspects of neutrophil, monocyte and APC function in the newborn. We will also highlight correlations between innate immune function and clinical diseases of newborns and infants.
Erythema toxicum neonatorum
A common, transient and benign rash of the newborn, characterized by raised lesions on an erythematous base. Recent evidence reveals that erythema toxicum probably reflects an innate immune response to initial skin colonization by Gram-positive bacteria (for example, coagulase-negative staphylococci) that penetrate neonatal skin through hair follicles. This bacterial penetration is associated with the activation of tissue macrophages and production of interleukin-1 (IL-1) and IL-6, which are capable of contributing to a systemic acute-phase response.

Neonatal skin: fragile, yet primed for defence. Neonatal skin is fragile at birth and even small breaks in the integrity of the skin can serve as lead points for infection25. The vernix caseosa, a waxy coating on newborns that is secreted by fetal sebaceous glands, contains APPs, including lysozyme, -defensins (also known as HNP1HNP3), ubiquitin and psoriasin, as well as antimicrobial free fatty acids that can act in synergy with APPs to kill microorganisms26. Accordingly, extracts of vernix caseosa possess antimicrobial activity in vitro against various bacteria and fungi, including Escherichia coli and Candida albicans2629. In addition, the skin from embryonic and newborn mice, as well as human newborn foreskin, shows a robust expression by epithelial cells of cathelicidin and -defensin antimicrobial peptides that exceeds that of adult skin, with synergistic antimicrobial activity against group B Streptococcus30. Overall, it is evident that the newborn is initially covered with a surface microbicidal shield to protect it during its transition to extra-uterine life27. Soon after birth, ~50% of all newborns develop a prominent but transitory rash, characterized by small erythematous papules often surrounded by a diffuse erythematous halo, known as erythema toxicum neonatorum. Emerging evidence indicates that erythema toxicum neonatorum is caused by reaction of neonatal skin to commensal flora, in particular Gram-positive staphylococci that colonize the skin and often penetrate into hair follicles. Such bacterial skin penetration may activate local macrophages to produce IL-6, (a multi-functional cytokine, production of which increases in the first days after birth31,32), which is likely to contribute to the postnatal systemic acute-phase response, including an increase in body temperature4,31 (FIG. 2a,b). Such early microbial exposure and the resulting innate immune response is likely to be important for maturation of the neonatal immune system. Newborn intestinal tract: colonization, tolerance and modulation . The fetal intestinal tract is normally bathed in sterile amniotic fluid that has been swallowed. However, upon delivery there is a rapid transition to primary colonization of the neonatal intestinal tract, presenting a crucial challenge for the immune system of the newborn (FIG. 2c). With a growing appreciation of the role of TLR signalling in the gut33, characterization of the development of TLR-mediated innate immunity in the fetal and newborn intestinal tract has been an area of recent focus. Enterocytes in the small intestine of the human fetus express basolateral TLR2 and TLR4 at 1821 weeks of gestation34. Studies of mouse and human tissue cultures show that fetal intestinal villous and crypt epithelial cells express TLR4 and MD2, key components of the lipopolysaccharide (LPS) receptor35. Stimulation of fetal intestinal cells with LPS resulted in higher levels of nuclear factor-B (NF-B) activation and production of CXC-chemokine ligand 8 (CXCL8) and CXCL2, compared with adult intestinal epithelial cells36,37 (for more on neonatal chemokines see BOX 2). However, such robust intestinal epithelial inflammatory

Anatomical considerations In general, the tasks of the innate immune system are to shield the body from microbial invasion, by reducing the number and virulence of microorganisms, and to coordinate and instruct the adaptive immune response. The function of fetal and neonatal innate immunity can be understood in relation to the anatomical sites at which it has a sentinel role in protecting the fetus. Therefore, the uterus must be kept sterile and, to this end, it expresses robust antimicrobial defence mechanisms (BOX 1; FIG. 1). Following birth, the neonatal skin and gut are rapidly colonized with microbial flora. Accordingly, birth is characterized by a distinct expression pattern of innate immune molecules by the skin and mucosal epithelia, and by the mobilization of an acute-phase response in peripheral blood, all of which are thought to be aimed at preventing infection while avoiding excessive inflammatory responses to microbial products.

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Antimicrobial proteins and peptides PLA2 Lactoferrin Defensins BPI Cathelicidin Histones

Syncytiotrophoblast Cytotrophoblast Histone

Amniotic fluid Cervical plug Umbilical cord

Skin epithelium

Blood
BPI

Defensins

Cathelicidin

Blood neutrophil

Figure 1 | Expression of antimicrobial proteins and peptides (APPs) in utero. The cervical plug, which contains many APPs, including lactoferrin and -defensin peptides, serves to separate the vagina, which is normally colonized with multiple microorganisms, from the normally sterile intra-uterine compartment. The maternal fetal membranes of the placenta secrete various APPs into the amniotic fluid, including lactoferrin, bactericidal/permeability-increasing protein (BPI), histones, phospholipase A2 (PLA2) and -defensins. The fetus itself has altered expression of APPs: whereas expression of cathelicidin and -defensin peptides by skin epithelium is increased, cellular concentrations of BPI in neutrophils are reduced.

of necrotizing enterocolitis37,41, an intestinal disease that occurs most frequently in pre-term newborns42. Thus, the failure of pre-term neonates to appropriately downregulate responses to LPS seems to significantly contribute to their susceptibility to necrotizing enterocolitis. Although it is currently unclear what role(s) intestinal TLR4 may have in utero, its distinct functional expression at birth clearly has important roles in neonatal intestinal health and disease. Neonatal intestinal immunity can be significantly modified by breastfeeding, and it is hypothesized that the overall effect of breast-milk-mediated intestinal immunomodulation results in sub-clinical infections that gradually stimulate immunological memory to pathogens while reducing inflammation43. Breast milk contains diverse immunological factors, including innate immune molecules such as the APPs, lactoferrin and lysozyme8,44,45. Human breast milk also contains factors that can modulate TLR signalling, including soluble TLR2, which can competitively inhibit signalling through membrane TLR2 (REF. 46), as well as an ~80 kDa protein that, by an as-yet-unknown mechanism, inhibits TLR2-mediated and activates TLR4-mediated transcriptional responses in human intestinal epithelial and mononuclear cells47. It has been speculated that reduced TLR2 reactivity at birth may facilitate the normal establishment of beneficial Gram-positive bifidobacteria intestinal flora. Newborns fed breast milk that contains relatively low levels of soluble CD14, a protein that mediates LPS signalling in the absence of membranebound CD14, have a higher risk of subsequent allergy, which has been interpreted as being consistent with the hygiene hypothesis48. Therefore, enhanced responses to endotoxin might be associated with a diminished risk of subsequent atopy. The neonatal respiratory tract: TLR and APP expression. Mouse studies have shown that expression of TLR2 and TLR4 is almost undetectable in the lungs of an immature fetus (gestational days 1415, which is the equivalent of ~30 weeks of gestation in humans), but increases several-fold during prenatal development and after birth49. There is also evidence of post-natal impairment of TLR2 and TLR4 expression in a rat model, as intrapulmonary inflammatory responses, including leukocyte recruitment, following intratracheal administration of LPS or Gram-negative bacteria are impaired early in life and do not approach adult levels until approximately 4 weeks of age50. Of note, exposure of epithelia to TLR agonists (for example endotoxin) activates expression of APPs through the production of IL-1 and consequent induction of human -defensin-2 (HBD2)51. Therefore, expression of HBD2 (the predominant -defensin in the human neonatal lung) by the respiratory epithelium is developmentally regulated and inducible by IL-1 in a myeloid differentiation primary-response gene 88 (MyD88)-dependent fashion52. Accordingly, the abundance of HBD2 in human neonatal tracheal aspirates increases with increasing gestational age. Of note, age-dependent susceptibility of newborn piglets to Bordetella pertussis bronchopneumonia correlates

Crypts
Tubular invaginations of the intestinal epithelium. Paneth cells are found at the base of the crypts and produce antimicrobial proteins and peptides, including phospholipase A2 and defensins, as well as stem cells, which continuously divide and are the source of all intestinal epithelial cells. Villi are projections into the lumen and have an outer layer of cells that mainly consists of mature, absorptive enterocytes but also contain mucus-secreting goblet cells.

responses to LPS could pose a serious danger shortly after birth, when the newborn is rapidly colonized by intestinal flora, including LPS-bearing Gram-negative bacteria38. Accordingly, exposure of perinatal intestinal cells to LPS has recently been shown to result in the loss of intestinal-epithelial-cell responsiveness to LPS, which was associated with downregulated expression of IL-1-receptor-associated kinase 1 (IRAK1), an essential intermediary for epithelial TLR4 signalling36 (FIG. 2c). This postnatal endotoxin tolerance may facilitate the adaptation of the newborn to subsequent microbial colonization and to support hostmicrobe homeostasis that is required for commensal interactions39. In parallel, the development of Paneth cells (which are a rich source of APPs) in the small intestine of newborns contributes to the clearance of bacteria such as E. coli40. The developmental expression of neonatal intestinal innate immunity is of substantial interest given the emerging evidence for an important role of endotoxin and intestinal TLR4 expression in the development

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a

Gram-positive bacteria

Erythema toxicum rash Liver

Epidermis

Intestine Hair shaft

Dermis

c
IL-1 Skin macrophage LPS IL-6 Intestinal lumen Gram-negative bacteria

To portal circulation (liver)

b
IL-1R IL-6R Acutephase proteins

Intestinal epithelial cell MD2 MyD88 P IB p65 p50 NF-B CXCL2 TLR4 IRAK4 IRAK1

C/EBP

Hepatocyte

Hepatic vein

CRP

MBL

LBP

Soluble CD14

Necrotizing enterocolitis
(NEC). A gastrointestinal disease predominantly affecting premature low-birthweight infants. NEC involves infection and inflammation that causes destruction of the intestine. Although the pathophysiology of NEC is not yet completely defined, increasing evidence indicates that immaturity of intestinal innate immune function of the premature gut, characterized by over-exuberant interleukin-8 responses of intestinal epithelial cells to lipopolysaccharide, is a major factor.

Figure 2 | Postnatal microbial colonization and the acute-phase response. Shortly after birth the newborn is colonized by microbial flora. a | Neonatal skin is colonized by Gram-positive bacteria (for example, coagulase-negative staphylococci) that often gain access to the skin through hair follicles and induce a benign rash known as erythema toxicum. At sites of eythema toxicum rash, neonatal macrophages produce interleukin-1 (IL-1) and IL-6. b | IL-1 and IL-6, through activation of the transcription factor CCAAT/enhancer-binding protein- (C/EBP), can contribute to the acute-phase response, triggering hepatocyte production of plasma proteins, such as C-reactive protein (CRP), lipopolysaccharide (LPS)-binding protein (LBP), soluble CD14 and mannose-binding lectin (MBL), which have roles in the clearance and detoxification of microbes and microbial toxins. c | At (or soon after) birth the intestinal tract of the newborn is first exposed to LPS that is derived from Gram-negative flora. Fetal (and pre-term neonatal) intestinal epithelial cells have markedly enhanced inflammatory responses to LPS. Mouse studies indicate that in full-term newborns, potentially harmful inflammatory responses to LPS are usually dampened by internalization of LPS by intestinal epithelial cells that induces downregulation of IL-1-receptor-associated kinase 1 (IRAK 1), thereby contributing to intestinal endotoxin tolerance. Such adaptations are central to the development of commensal relationships. CXCL2, CXCchemokine ligand 2; IB, inhibitor of nuclear factor-B; MyD88, myeloid differentiation primary-response gene 88.

with developmentally regulated upper respiratory tract expression of the porcine -defensin 1 (pBD1) and exogenous replacement with pBD1 protects against B. pertussis infection53. The relevance of neonatal respiratory innate immunity to the (patho)physiology of the newborn is beginning to emerge. Chorioamnionitis is an infection of the placental membranes and amniotic fluid by bacteria that express TLR agonists such as E. coli and group B

Streptococcus. Chorioamnionitis induces the production of IL-6, a cytokine that enhances fetal-lung branching morphogenesis, possibly providing a mechanism by which the innate immune system can hasten the development of respiratory function in the context of infection-induced pre-term birth54. Of note, TLR-mediated responses can be modulated by respiratory infection. For example, Ureaplasma urealyticum, a mycoplasmal organism that can colonize the neonatal respiratory tract

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Box 2 | The role of chemokines in the development of neonatal immunity
Chemokines and antimicrobial peptides are overlapping categories of host defence molecules, as many chemoattractant cytokines often also possess direct antimicrobial activity. In addition, many molecules that were originally characterized as antimicrobial peptides are now known to also possess chemoattractant and other immunomodulatory activities152,153. As such, chemokines and antimicrobial peptides are an important link between innate and adaptive immunity. Of note, chemokines are expressed in an age-dependent manner, have a role as early as embryonic implantation154 and show gestational age-dependent expression155. Recent work in an ovine model showed that fetal expression of CC-chemokine ligand 25 (CCL25; expressed in the thymus and gut) and CCL28 (expressed in the large intestine, trachea, tonsils and mammary gland), and their receptors, which are expressed in the thymus and mucosal tissues, increase with gestational age156. By mediating lymphocyte colonization of fetal tissues, chemokines might, therefore, have important roles in the maturation of the fetal immune system. Chemokines are also necessary for post-natal localization of lymphoid progenitors to the thymus157 and secondary lymphoid tissues, including the mucosa-associated lymphoid tissues. In a mouse model, impaired neonatal pulmonary chemokine production corresponds to delayed clearance of Pneumocystis carinii pneumonia158. Pre-term human neonates have particularly low basal serum levels of the chemokine CCL5 (also known as RANTES)159. Influenza-virus-induced CCL3 production by fullterm human neonatal mononuclear cells is impaired160, and human newborns with relatively lower levels of CCL3 production have an increased risk of perinatal HIV transmission161. Certain chemokines, such as CXC-chemokine receptor 3 (CXCR3; also known as IP10), are upregulated during neonatal bacterial infection, showing potential use as diagnostic markers162.

Chorioamnionitis
Infection of the placental tissues and amniotic fluid as a result of ascending infection by bacteria that can be present in the vagina, such as Escherichia coli and group B Streptococcus. Chorioamnionitis can cause maternal bacteremia and can lead to pre-term delivery and neonatal infection.

Bronchopulmonary dysplasia
(BPD). A disease of the respiratory system that is most frequent in pre-term infants, and is characterized by inflammation and scarring of the lungs resulting in abnormal development of lung tissue. Recent evidence shows that Toll-like-receptor-mediated inflammatory responses can reproduce the pulmonary histological findings characteristic of BPD, raising the possibility that innate immune responses may contribute to its pathophysiology.

and is associated with pre-term birth and abnormal lung development55, has TLR2 and TLR4 agonist activity56, and induces the production of TNF, both directly and in synergy with LPS, from pre-term neonatal monocytes57. Thus, this common pathogen may have important pathophysiological roles. Moreover, injection of LPS into the amniotic fluid of fetal mice at day 15 when TLR4 expression first appears induces, over a 2-day span, TLR4-mediated inflammatory responses that alter airway fibronectin expression, potentially inhibiting distal-airway branching and alveolarization58. In addition, activation of TLR2 or TLR4 in the developing mouse lung inhibits the production of fibroblast growth factor 10, thereby disrupting normal myofibroblast positioning in lung development59. Overall, these observations potentially link innate immune responses with the abnormal fetal lung development that is characteristic of bronchopulmonary dysplasia. The respiratory tract is an important route of exposure for environmental adjuvants (aeroadjuvants) and antigens (aeroallergens), and therefore has an important role in modulating the balance of immune maturation. Age-dependent changes in the innate immune response of the respiratory tract are, therefore, likely to also be relevant for the development of allergy. A seminal study revealed that in nasal mucosa explants from atopic children, but not atopic adults, LPS enhances allergen-induced T-cell reactivity and/or proliferation, the production of TH1-cell-polarizing cytokines, IL-10 production and TLR4 expression by lymphocytes60. Therefore, in young children, LPS might inhibit allergic inflammation by skewing local immune responses from a TH2-cell to a TH1-cell response. Children who have environmental exposure to muramic acids and/or

peptidoglycans (TLR2 agonists) have reduced risk of wheezing (which is a symptom of reactive airways that is predictive of asthma severity)61. Sterile house-dust extracts have MyD88-dependent adjuvant activity, which is TH2-cell polarizing when given as a weekly intranasal vaccination (along with ovalbumin antigen) but is actually tolerogenic when given daily as a low-dose intranasal exposure16. From an epidemiological perspective, chronic exposure over the first 3 months of life to farming, endotoxin contained in house dust (a TLR4 agonist) and household pets (such as cats and dogs) are all associated with an enhanced ability of neonatal and infant peripheral-blood cells to produce the TH1-cellpolarizing cytokine interferon- (IFN) in response to phorbol myristate acetate and concanavalin A62. These observations indicate that certain patterns (for example, dose, frequency or route) of exposure to microbial TLR agonists early in life accelerate the maturation of the TH1-cell response and protect against allergic and atopic diseases, which is the essence of the hygiene hypothesis18,63. Of note, it is increasingly recognized that CD4+CD25+ regulatory T (TReg) cells, which inhibit TH1-cell immunity thereby maintaining peripheral T-cell tolerance, are particularly abundant and potent at birth64. Moreover, TLR activation can enhance TH1-type responses by reversing TReg-cell function by indirect effects (through dendritic cell (DC)-derived IL-6)65 or by direct effects (through activation of TLR8 on TReg cells)66. Future work aimed at characterizing the developmental expression of TLR-mediated effects on neonatal and infant TReg cells will be of substantial interest in further exploring and refining the hygiene hypothesis. Overall, the innate immune mechanisms of the neonatal respiratory tract provide protection against microbial infection and prevent over-exuberant inflammation, while mediating the effects of environmental TLR agonists that serve as aeroadjuvants to reduce the atopic potential of aeroallergens. Neonatal blood plasma: adenosine and acute-phase products. The levels of multiple soluble plasma proteins that have a role in innate immunity are lower in newborns than in adults. Neonatal plasma concentrations of complement components are diminished compared with those in adults, ranging from ~1070% of adult levels8. A deficiency in complement might contribute to the inability of newborns to limit the replication of many bacterial strains in the blood67 and, as complement components also have a role in adaptive immunity68, it might contribute to the impairment of neonatal adaptive responses69. The metabolic state of the newborn, initially characterized by low oxygen tension (that can be further exacerbated by prolonged labour, uterine contractions and/or fetal distress), modulates neonatal plasma concentrations of early-response cytokines. Such hypoxia is known to increase the production of IL-6 and IL-8 (REF. 70) , cytokines with important roles in labour, while limiting production of TNF 71. Endogenous mediators are also elevated during hypoxia and/or stress. Adenosine, an endogenous purine metabolite

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for which concentrations rise during hypoxia, has immunomodulatory properties 72 and is found at relatively high concentrations in neonatal cord-blood plasma where it inhibits TLR2-mediated TNF production through the activation of A3 adenosine receptors on neonatal mononuclear cells71 (discussed in greater detail later). Plasma concentrations of multiple acute-phase products that are derived from the liver change dramatically during the first days of life, probably reflecting initial exposure of the newborn to microbes and microbe-derived TLR agonists. Histological analysis of erythema toxicum neonatorum lesions from human newborns during the first week of life showed that skin macrophages ingest bacteria and produce IL-1 (REF. 73), and studies of neonatal blood plasma during this same time frame showed rising plasma levels of IL-6, exceeding basal adult blood plasma IL-6 levels12 (FIG. 2a). Both IL-1 and IL-6 can trigger acute-phase responses by activating hepatocytes to synthesize and secrete an array of acute-phase proteins. These include mannose-binding lectin (MBL) 74, soluble CD14 (REF. 75) , C-reactive protein (CRP) and LPS-binding protein (LBP)12, all of which are initially low at birth but rise during the first week of life (FIG. 2b). Mouse studies have begun to define the transcriptional control of the neonatal acute-phase response, in that LPS- and IL-1-induced transcription of genes encoding acute-phase proteins occurs through the transcription factor CCAAT/enhancer-binding protein- (C/EBP) and binding of signal transducer and activator of transcription 3 (STAT3) to cognate promoter sequences76 (FIG. 2b). Conversely, plasma concentrations of negative acute-phase plasma glycoproteins such as prealbumin31 and fetuin, which suppresses TNF production but preserves IL-6 production77, are initially high at birth, then drop7880. Given the role of multiple acute-phase reactants, including LBP and soluble CD14 (which, at high or acute-phase concentrations actually inhibit LPS activity)81, in the clearance and/or detoxification of LPS and other TLR agonists, one might speculate that the rapid activation of the acute-phase response soon after birth may be directed at clearance of any microbial products that have translocated across mucous membranes during birth and/or initial colonization in order to avoid excessive inflammation as the newborn first meets the outside world. and function of complement receptor 3 (CR3; also known as CD11b/CD18)85 and diminished expression of L-selectin 86. In general, as with other aspects of immune function, these neutrophil defects are even more pronounced with prematurity, but begin to correct within the first weeks of life82. Impaired recruitment of neonatal neutrophils to inflammatory sites might be related not only to diminished integrin and selectin expression, but also to the propensity of neonatal monocytes and APCs to produce relatively large amounts of IL-6 (REFS 12,87), a cytokine that inhibits neutrophil migration to inflammatory sites88. Neonatal neutrophils also exhibit impairments in microbicidal mechanisms. These neutrophils contain reduced amounts of some APPs, including lactoferrin (~50% of adult levels)89 and BPI (~30% of adult levels)90, but normal amounts of defensin peptides90. Reduced expression of BPI correlated with diminished activity of neonatal neutrophil extracts against Gram-negative bacteria90. Neutrophils of pre-term newborns show impaired upregulation of oxidase activity in response to staphylococci, which are common neonatal pathogens91. LPS-induced priming of neutrophils for enhanced oxidase activity (in response to a second stimulus) is also impaired in neutrophils from newborns, possibly owing to diminished upregulation of membrane CD14 that normally contributes to enhancement of LPS signalling92. Compartmentalization of the protein tyrosine kinase p53/56lyn in a membrane-granule fraction of neonatal neutrophils, as opposed to its cytosolic localization in neutrophils from adults, may limit its mobility and thereby diminish LPS priming of neutrophils from newborns93. Overall, neonatal defects in the amplification, mobilization and function of the neutrophil response have clinical relevance in that neutropaenia is a welldescribed and ominous finding in newborns presenting with bacterial sepsis1. Monocytes and APCs. There are both quantitative and qualitative differences between monocytes and APCs from newborns and adults. Qualitative differences in monocytes are evident in utero, as third-trimester phenotyping of fetal or neonatal circulating monocytes by flow cytometry reveals that human fetal monocytes express reduced levels of MHC class II molecules, which potentially contributes to impaired APC activity94. Mouse studies show that the neonatal DC system is immature, and includes a distinct population of splenic DCs (having a higher ratio of CD4CD8+ DCs to CD4+CD8 DCs compared with the adult DC system) and impaired in vitro production of IL-12 p70 in response to a CpG oligodeoxynucleotide motif (ODN 1668), a pattern that normalized at ~5 weeks of age95. However, this defect in APC activity may be stimulus-specific, in that a different CpG motif (ODN 1826) stimulated CD11c+ splenic DCs purified from 7-day-old newborn mice to produce at least as much IL-12 p70 as their adult counterparts96. Mouse CD5+ B cells may contribute to the impairment of neonatal APC function by TLR9-mediated IL-10 production, which inhibits IL-12 synthesis by neonatal DCs97. Overall, reduced TH1-cell-polarizing fetal APC activity is
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Neonatal leukocytes Neutrophils: quantitative and qualitative defects. Newborn mammals have a reduced number of quiescent granulocyte and monocyte progenitor cells and a diminished precursor storage pool that can result in a quantitative defect in neutrophil numbers during stress conditions (for example, in sepsis). From a qualitative standpoint, neonatal neutrophils show impairment of multiple functional aspects, including chemotaxis, rolling adhesion, transmigration and lamellipodia formation8284. Such functional defects correlate with a relatively high proportion of immature neutrophils in umbilical cord blood that show reduced expression
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thought to be necessary to reduce the risk of alloimmune reactions between mother and fetus, and phenotypic and stimulus-specific functional immaturities are present at birth in both mice and humans14,95,98100. The potential relevance of TLR expression in neonatal health and disease includes the recent demonstration that TLRs participate in the recognition of microbial pathogens that are relevant to neonates, including pathogens recognized by TLR2 (group B Streptococcus84, Listeria monocytogenes, Mycoplasma hominis101, C. albicans hyphae102 and cytomegalovirus102), TLR4 (Enterobactericeae, C. albicans blastoconidia102, and respiratory syncitial virus103) and, in mice, TLR11 (Toxoplasma gondii)104. In this context, the functional maturation of TLR expression has been of substantial interest. Cord-blood monocytes of pre-term humans (2537 weeks) have diminished basal TLR4 expression but normal basal TLR2 expression compared with adult peripheral-blood monocytes105. Several studies have established that cord-blood monocytes that are derived from full-term human newborns express comparable amounts of TLRs to adult monocytes75,106. Although basal TLR expression of full-term neonatal blood monocytes is similar to that of adults, the functional consequences of neonatal TLR activation are very different. It has been appreciated for some time that, despite the presence of higher concentrations of monocytes at birth, the addition of LPS to whole cord blood from human newborns results in diminished production of TNF compared with adult peripheral blood107. Indeed, newborn-derived monocytes cultured in whole blood or purified and cultured in autologous (newborn) blood plasma show a 13-log impairment in TNF production in response to agonists of TLR1TLR7 (REFS 12,71,75). Human neonatal APCs also have impaired production of type I IFNs (that is, IFN and IFN), including impaired IFN production by neonatal plasmacytoid DCs in response to polyinosinicpolycytidylic acid (polyI:C; a TLR3 agonist) and CpG (a TLR9 agoinst)108 and impaired IFN production by neonatal monocyte-derived DCs in response to LPS (a TLR4 agonist)109. Similarly, CpG ODN fails to fully overcome the bias of neonatal mononuclear cells towards the production of the TH2-cell-polarizing cytokine IL-13 (REF. 110). Such a pattern is not confined to microbial TLR agonists: upon phagocytosis of necrotic cells, DCs from neonates show impaired production of TNF, upregulation of co-stimulatory molecules (CD80 and CD86) and stimulation of T-cell proliferation when compared with DCs from adults111. Neonatal mice injected subcutaneously with LPS show impaired IFN production and delayed kinetics, but a greater peak, of TNF levels, as well as impaired staphylococcal enterotoxin B (now known to be a TLR2 agonist112)-induced TNF production113. This and other studies indicate that neonatal monocytes and APCs may have a greater impairment in TLR2-mediated, rather than in TLR4-mediated, TNF production47,71. Overall, upon birth, neonatal APCs manifest selective impairments in the production of TH1-type immune responses following innate immune recognition of multiple exogenous and endogenous stimuli. By contrast, TLR-mediated production of IL-6, IL-10 and IL-23 by neonatal monocytes, macrophages and myeloid DCs is actually enhanced relative to adult cells1214 (TABLE 1). Robust production of IL-23 by human neonatal DCs is noteworthy in that it increases expression of IL-17 (REF. 13), a cytokine that can induce epithelial antimicrobial peptides114, and expression of these

Table 1 | Polarization of TLR-mediated cytokine responses of neonatal cord-blood-derived monocytes and antigen-presenting cells
Cytokine Relative expression in newborns

Effect of cAMP on cytokine production


General function

Comment

References

TNF IFN IFN IL-12 IL-1 IL-6

Pro-inflammatory; activates neutrophils; TH1-cell response Antiviral; contributes to vaccine responses Activation of macrophages; induction of IL-12; TH1-cell response p40p35 heterodimer activates cellmediated immunity; TH1-cell response Endothelial adhesion; fever; acute-phase response Acute-phase response; inhibits tissue neutrophilia; inhibits TReg cells and promotes TH17 cells Neutrophil chemoattractant Anti-inflammatory; inhibits TNF, IL-1 and IFN production p40p19 heterodimer promotes TH17 cells

TNF associated with spontaneous abortion and pre-term labour Important for MHC class I expression Newborns have impaired killing of intracellular pathogens Neonatal defect in p35 promoter nucleosome remodelling Newborn febrile response is blunted May contribute to acute-phase response at birth Expression increased during hypoxia; role in parturition Blocking IL-10 can restore IL-1 production IL-17 enhances epithelial expression of antimicrobial peptides

71 108,163 11 126 164 12,165

IL-8 IL-10 IL-23

166 14,167 13,125

, increased; , decreased; , unchanged; cAMP, cyclic AMP; IFN, interferon; IL, interleukin; TH, T helper; TLR, Toll-like receptor; TNF, tumour-necrosis factor; TReg, T regulatory.

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peptides is elevated at birth30. Importantly, the neonatal bias away from TH1-cell-polarizing cytokine responses, as reflected by a high ratio of IL-6 to TNF production by mononuclear cells, is not only evident in the response to individual TLR agonists12, but also to whole commensal bacteria5 and to herpes simplex virus115 in vitro. In line with these observations, a recent mouse model of neonatal sepsis produced by generalized peritonitis (through cecal slurry administration) showed markedly diminished neonatal plasma TNF levels (<10% of adult levels), but enhanced neonatal IL-6 production116. Moreover, the bias of neonatal cytokine responses in favour of IL-6 might also be relevant in vivo in human newborns, as shown by a rising basal serum ratio of IL-6 to TNF, and of IL-6-inducible acute-phase reactants CRP and LBP, during the first week of life12. Overall, the IL-6induced acute-phase response induced by birth, together with a preserved IL-23IL-17 axis, can be seen as mobilizing both an external (mucosal and/or epithelial) and internal (blood plasma) shield of anti-infective proteins and peptides to protect the newborn against the risks of infection upon initial microbial colonization. The explanation of the distinct bias in neonatal cytokine production relates to the potentially negative consequences of excessive production of pro-inflammatory cytokines such as TNF, including associations with intra-uterine growth restriction117 and spontaneous abortion10. In addition, emerging evidence indicates that TLR-mediated pro-inflammatory cytokine induction by microglia might contribute to central nervous system damage in pre-term newborns that can culminate in cerebral palsy118,119, providing further rationale for this pattern of neonatal cytokine responses. Although such polarization is therefore beneficial, it also comes at a significant cost, leaving neonates particularly sensitive to infection with a broad array of microorganisms, including intracellular pathogens requiring efficient pro-inflammatory TH1-cell responses for clearance, such as L. monocytogenes and herpes simplex virus, among others120. Although many studies have shown a distinct pattern of neonatal cytokine production by monocytes and/or APCs, few have addressed the mechanisms for such differences as compared with adults. Recent evidence

Adenosine TLR A3 receptor APC TRIF

b MyD88
IRAK4 TRAF6

cAMP

PKA

IB p50 p65

TBK1

CREB P CREB p50 p65 IRF3

P CREB CBP CRE IL-6 p50 p65 Reduced TH1 pro-inflammatory cytokine production

c
IL-I2 p35

d
IRF3

IRF3 CBP IFN

Cerebral palsy
A disorder of posture and movement due to damage to motor areas of the brain, often associated with a history of perinatal complications. The pathophysiology of cerebral palsy is still under investigation, but recent evidence indicates that a combination of hypoxic ischemic insult and Toll-likereceptor-mediated inflammation can synergistically trigger neurodegeneration.

Figure 3 | Mechanisms for distinct function of human neonatal monocytes and antigen-presenting cells. a | High concentrations of adenosine, an endogenous immunomodulatory purine metabolite, in neonatal blood plasma act through adenosine A3 receptors on neonatal mononuclear cells to induce high (~20-fold greater than adult levels) intracellular concentrations of cyclic AMP (cAMP). cAMP is a secondary messenger that, through both protein kinase A (PKA)-dependent and PKAindependent pathways, can inhibit Toll-like receptor 2 (TLR2)-mediated tumour-necrosis factor (TNF) production while preserving production of interleukin-6 (IL-6). b | Neonatal monocytes have diminished expression of myeloid differentiation primary-response gene 88 (MyD88), a key adaptor molecule for TLR-mediated signalling. c | Failure of nucleosome remodelling of the Il12p35 gene promoter contributes to diminished TLR-mediated IL-12 p35 production by neonatal dendritic cells (DCs), an example of distinct regulation of neonatal cytokine production at the chromatin level. d | Lipopolysaccharide (LPS)-induced association of interferon (IFN)-regulatory factor 3 (IRF3) with cAMP-responsiveelement-binding protein (CREB)-binding protein (CBP) and IRF3 binding of DNA are reduced in human neonatal monocyte-derived DCs, resulting in impaired expression of IFN. AP1, activator protein 1; NF-B, nuclear factor-B; TBK1, TANK-binding kinase 1; TRAF, TNFR-associated factor; TRIF, TIR-domain-containing adaptor protein inducing IFN.

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indicates that the distinct physiological state of neonatal mononuclear cells at birth, including their exposure to distinct humoral factors inherent to gestation and/or birth, may substantially contribute to the polarization of their TLR-mediated cytokine responses. Of note, human neonatal cord-blood mononuclear cells contain ~20-fold greater intracellular concentrations of the second messenger cyclic AMP (cAMP)71, a molecule that can act through both a protein kinase A (PKA)-dependent and PKA-independent pathway to inhibit the stimulus (for example LPS)-induced production of TNF72 (FIG. 3a). cAMP may reduce TNF production through the inhibition of phosphorylation of p38 mitogen-activated protein kinase (MAPK)121, a cytosolic signalling intermediate for which activation is crucial for LPS-induced TNF production122; the phosphorylation of p38 is impaired in cord-blood monocytes from term and pre-term human newborns123,124. Intriguingly, cAMP is also known to inhibit the production of several other cytokines for which production is impaired in newborns (such as IFN, IFN and IL-12), while preserving or enhancing the expression of cytokines that neonatal monocytes and APCs produce in abundance (for example IL-6, IL-10 and IL-23)125. Therefore, the elevated intracellular cAMP concentrations of neonatal cells might provide a general physiological mechanism that contributes to the polarization of TLR-mediated cytokine responses by neonatal monocytes13,71 (TABLE 1). This cAMP hypothesis remains to be proven, and the postnatal duration of the high cytosolic cAMP content of neonatal mononuclear cells remains to be defined. Nevertheless, even if limited in effect to the immediate perinatal period, such cAMP-mediated polarization during initial exposure to microbes (including maternal-derived urinary and enteric microorganisms) and antigens might have profound effects on early immune responses. Additional mechanisms, which are not mutually exclusive, for the polarization of neonatal TLR-mediated responses have been identified. Although human neonatal cord-blood monocytes contain normal levels of mRNA of the TLR adaptor molecule MyD88 (REF. 75), expression of the MyD88 protein is reduced, possibly contributing to impaired TLR-mediated TNF production106,124 (FIG. 3b). Of interest, regulation of neonatal APC cytokine responses also occurs downstream of cytosolic signalling pathways, in that regulation of IL-12 expression by human neonatal monocyte-derived DCs is limited by a defect in nucleosome remodelling126 (FIG. 3c). Although many aspects of transcription-factor binding to relevant cis-acting elements of the IL-12 p35 promoter are similar in DCs from adults and newborns, chromatin-accessibility assays reveal that LPS-induced nucleosome remodelling, required for effective functioning of the upstream SP1 transcription factor sites, is substantially impaired in neonatal DCs. Therefore, neonatal IL-12 p35 gene transcription is repressed at the chromatin level. Of note, administration of IFN, possibly acting through IFN-activated transcription factors, restored both nucleosome remodelling and IL-12 p35 gene transcription in vitro, indicating that the modulation of nucleosome remodelling is central to efficient activation of neonatal APCs126. Impaired LPS-induced production of IFN by neonatal monocyte-derived DCs is associated with impaired interaction of IFN-regulatory factor 3 (IRF3) with cAMP-responsive-element-binding protein (CREB)-binding protein (CBP)109 (FIG. 3d). There are some exceptions to the general neonatal impairment in TH1-cell-polarizing responses to microbial agonists. Although neonatal monocytes and APCs show impaired production of T H 1-cell-polarizing cytokines to agonists of TLR1TLR7 (REF. 75), agonists of TLR8 (or TLR7 and TLR8), such as small antiviral imidazoquinoline compounds that are purine analogues and single-stranded viral RNAs, induce robust (comparable to adults) production of TNF and IL-12/IL-23 p40 by these cells, as well as upregulation of the expression of CD40 by neonatal myeloid DCs75,100. These strong adjuvant effects correlate with the ability of agonists of TLR8 (or TLR7 and TLR8) to robustly induce p38 MAPK phosphorylation and prolong the degradation of IB (inhibitor of NF-B)100. Similarly, certain CpG ODNs can enhance neonatal TH1-cell responses127, and are protective against neonatal lethality in a mouse model when co-administered as an immunostimulatory agent at the time of neurotropic Tacaribe arenavirus infection128. Whole group B streptococci, through robust activation of the alternative complement pathway and engagement of integrin-based complement receptors, induce marked TNF production by neonatal cord-blood monocytes that is equivalent to that of adult monocytes129. Mycobacterium bovis bacillus CalmetteGurin (BCG) is an example of one of the few vaccines that are active at birth and can induce a strong TH1-cell-polarizing cytokine response, including the expression of IFN, by CD4+ T cells in response to a mycobacterial purifiedprotein derivative (PPD)130. These examples indicate that stimuli with certain characteristics, including the ability to effectively activate certain TLR pathways and/or the complement system, are able to overcome neonatal impairments and induce robust production of TH1-cellpolarizing cytokines. The mechanisms, teleology and potential translational implications for preservation of neonatal TH1-cell responses to such stimuli remain to be fully explored.

Future prospects From a basic standpoint, much remains to be learned about the distinct functional expression and agedependent maturation of innate immune molecules at birth. Unravelling the relationships between distinct aspects of neonatal humoral and cellular immunity, including the impact of physiological mediators on neonatal cellular responses, will enhance our mechanistic understanding of the innate immune system of the newborn. The microbiological and immunological events during the first days of life, including establishment of a commensal gut flora, probably impact on health, infection and allergy. What are the implications of the acute-phase response that is initiated at birth? What impact does this have on the newborns first responses to antigens and microbes? How do
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such responses affect adaptive immunity? The impact of genetic variation in innate immune molecules on susceptibility of newborns to infection will also be of interest131. Owing to the important role of innate immunity in neonatal health and disease, the intense biopharmaceutical development of molecules that are derived from or that modulate the innate immune system, including antimicrobial proteins and peptides and TLR agonists, is likely to have clinical relevance to neonatal medicine. Given the limited manner (that is, the limited range of symptoms) by which neonates manifest a broad array of diseases, one area of unmet medical need is the development of efficient diagnostic markers that can distinguish infection from other causes of neonatal inflammation and/or clinical deterioration132. The acute-phase reactant CRP is already used in this context, and additional innate immune markers, including surface expression of neutrophil TLRs133, are also under evaluation for this purpose. Clinical trials of granulocyte/monocyte colonystimulating factor (GM-CSF) to enhance the quantity and quality of neonatal neutrophils and monocytes have not yet shown a significant clinical benefit, but further clinical evaluation focusing on a subpopulation of pre-term newborns at high risk for severe neutropaenia is on-going134. The evaluation of recombinant APPs as adjunctive therapy for neonatal infection is also proceeding23. TLR agonists might represent tools to enhance the defence against microorganisms128,135,136 or to shift innate immune responses of neonatal APCs away from the production of TH2-cell-polarizing cytokines, thereby potentially reducing allergy24. Finally, as birth is a relatively reliable point of contact with health-care systems worldwide, and therefore vaccines given at birth reach a relatively high proportion of the population137, the potential of certain TLR agonists capable of efficiently activating TH1-cell-polarizing responses from neonatal APCs are of substantial interest as novel neonatal vaccine adjuvants69,100,138.

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Acknowledgments
I thank P. Elsbach, R. Geha, R. Munford, P. Pizzo, J. Weiss and M. Wessels for their mentorship. P. Bibbins created concept illustrations for FIGS 2,3. O.L.s laboratory is supported by the National Institutes of Health, a Dana Human Immunology Award and by XOMA (U.S.) L.L.C.

Competing interests statement


Among the funding sources to the authors laboratory is research support from XOMA (U.S.) L.L.C. that manufactures recombinant BPI.

DATABASES
The following terms in this article are linked online to: Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query. fcgi?db=gene IL-1 | IL-6 | IL-12 | MYD88 | TLR2 | TLR4 | TNF

FURTHER INFORMATION
Ofer Levys homepage: http://www.childrenshospital.org/ cfapps/research/data_admin/Site364/mainpageS364P0.html Access to this links box is available online.

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