The developing human preterm neonatal immune system: A case for more research in this area (original) (raw)

. Author manuscript; available in PMC: 2015 Sep 1.

Published in final edited form as: Clin Immunol. 2012 Aug 17;145(1):61–68. doi: 10.1016/j.clim.2012.08.006

Abstract

Neonates, particularly those born prematurely, are among the most vulnerable age group for morbidity and mortality due to infections. Immaturity of the innate immune system and a high need for invasive medical procedures in the context of a preterm birth make these infants highly susceptible to common neonatal pathogens. Preterm infants who survive may also suffer permanent disabilities due to organ damage resulting from either the infection itself or from the inflammatory response generated under an oxidative stress. Infections in preterm infants continue to pose important healthcare challenges. Yet, developmental maturation events in the innate immune system that underlie their excessively high vulnerability to infection remain largely understudied. In this review article, we identify pertinent knowledge gaps that must be filled in order to orient future translational research.

Keywords: Innate immunity, Infant, Premature, Toll-like receptors, Infection, Oxidative stress

1. Introduction

Neonates are one of the highest risk age groups for mortality and morbidity from infection. According to the World Health Organization, 3.7 million neonates less than 28 days of age died in 2010 – 37% of these deaths were due to infectious causes [1]. Among those, preterm infants are undoubtedly at highest risk and the health consequences that ensue are broad and serious [2]. Eleven percent of all infants are born premature (i.e. before 37 weeks of gestation), representing about 12.9 million infants born prematurely each year worldwide [3]. Unlike respiratory distress-related mortalities, which have shown a constant decline, the mortality from neonatal infection in preterm infants has increased over the last 20 years [4]. In North America, nearly one in six premature infant develops an invasive infection (i.e. bacteremia, pneumonia or meningitis) in their first weeks of life - many of which are fatal - and this risk is even higher in smallest preterm infants [5; 6; 7]. Unfortunately, neonatal infection can also cause irreversible damage to preterm infants’ organs (like their lungs, brain and intestines) also resulting in a greater risk of long-term neurodevelopmental impairment in survivors [8]. Finally, illnesses due to infection in this age group impose considerable pressure on health care resources, adding approximately twenty-five thousand of dollars per episode of infection per infant, out of the $25 billion annual economic impact of prematurity in the US alone [9; 10].

Preterm infants’ vulnerability to infection can be attributed to two main reasons: a frequent need for life-saving medical interventions that interfere with the body’s protective mucosal and epithelial barriers (e.g. mechanical ventilation, intravenous lines, etc.), as well as developmental immaturity leading to deficits of the immune system (e.g. lack of maternally transferred antibodies, which normally occur late in pregnancy, see below). A better understanding of human innate immune system at the very earliest stage in life is necessary in order to orient the development of safe, targeted drug therapies to improve health of these children. Yet, despite serious outcomes translational immunology research on the human newborn immune system remains limited. For example, at the time of writing of this manuscript, a search through the US National Institutes of Health public database (Pubmed) identified 905,094 publications related to the field of human immunology, of which 63,110 (7%) were concentrated on newborns and infants, whereas only a relatively minuscule proportion (2,413 publications, or <0.3%), concerned the premature infant immune system1. Detailed literature reviews of the neonatal [11] and particularly, the preterm immune system have been recently published [12]. Here, we present our perspective on the latest research in this area. We identify knowledge gaps as well as some of the emerging research areas on the characteristics of the innate immune system in infants born very early in gestation.

2. The developmentally immature preterm innate immune system

Protection against pathogens is achieved through the coordinated actions of the innate and the adaptive arms of the immune system. Newborns rely heavily on their innate immune defenses as their “educated” adaptive immunity fully develops only later, in the early years of life [13]. Infants born at term benefit from supplemental protection afforded by maternal antibodies transferred to them through the placenta (figure 1) [14]. Extreme preterm infants, however, substantially lack trans-placental transfer of maternal antibodies, which largely occurs during the third trimester of gestation [15; 16].

Figure 1.

Figure 1

Developmental changes occurring in the human immune system early in life. This figure illustrates maturational events occurring in major adaptive and innate immune functions as the human transitions from a fetal tolerance state and becomes exposed to microorganisms as well as other environmental antigens de novo after birth. Adaptive immune functions [top panel]: Maternal transplacental antibody transfer (IgG) mainly occurs during late gestation, followed by maternal antibody protection (IgA) acquired through breast-milk after birth. Infants’ own antibody response become fully mature later during early childhood. Neonatal T cells are largely biased towards helper type II responses and humans display high proportions of T regulatory and Natural Killer T cells at birth [91]. Innate immune functions [bottom panel]: Pro-inflammatory (IL-1β, IL-6, TNF-α, IL-12, IL-23) and anti-viral (IFN-α) cytokine responses are largely attenuated in preterm infants, whereas production of the anti-inflammatory IL-10 cytokine is relatively high during late gestation and at birth.

Even though the high vulnerability of preterm infants to infection cannot be ascribed to individual immune components, major functional differences exist when comparing to adults, term and preterm neonates (figure 1). Generally speaking, the composition of circulating white blood cells numerically differs among neonates of different gestations, likely reflecting dynamic developmental phases (figure 2). The proportions of both lymphoid and myeloid cells are generally higher in the peripheral blood of a healthy term neonate [17; 18; 19; 20]. Other major functional deficiencies in preterm innate immune functions have been recently reported include reduced Pattern-Recognition Receptor (PRR) function (discussed below), extracellular bacterial elimination [21] and leukocyte endothelial adhesive/rolling [22].

Figure 2.

Figure 2

Blood leukocyte count in preterm and term newborns and adults. Adapted from reference [18].

2.1 Attenuated Toll-like receptor function in preterm neonates

Innate immune surveillance is achieved through activation of cell surface receptors called Pattern Recognition Receptors (PRRs), which include Toll-like receptors (TLR), the Nucleotide Oligomerization Domain (NOD)-like receptors (NLR) and the retinoic acid-inducible gene I (RIG-I)-like receptors (RLR). TLRs have been a major focus of neonatal immunological research due to the extent of basic science knowledge in this area. Humans have ten known TLRs whose biological responses are dictated by the structural characteristics, cell-type specific expression and cellular localization profiles [23]. TLR cytokine responses are markedly attenuated in preterm cord blood compared to their term counterparts or to adults, when examined either in populations of cells or on a per-cell basis ([12] and below). Specifically, preterm neonates have significantly reduced pro-inflammatory cytokine (i.e. IL-1β, IL-6, TNF-α, figure 1) responses when stimulated with endotoxin (lipopolysaccharide, a potent TLR4 agonist) [24; 25; 26; 27; 28], IL-1 [29] or whole micro-organisms in vitro [30; 31; 32; 33]. They also produce limited amounts of anti-viral interferon-α even though their proportion of blood plasmacytoid dendritic cells, main source of this cytokine, is comparable to term neonates or adults [34]. In contrast, neonatal anti-inflammatory responses (e.g. IL-10 or TGF-β) are high, especially in preterm neonates at the lower end of gestation, although discrepancies exist among studies most likely due to methodological differences (e.g. sample size, stimulating conditions, etc) [27; 30; 31; 34; 35; 36] (figure 1). The attenuated cytokine responses observed in preterm neonates are also consistent with reduced protective antibody responses following administration of routine immunizations [37; 38].

2.2 Mechanisms of innate immune response attenuation

Some investigators have attributed part of the attenuation in pro-inflammatory cytokine responses to a gestational age-dependent reduction in surface expression of TLR4 and its co-receptor CD14 [39; 40; 41], as well as to reduced expression of MyD88 and IRF5 (two key components of the TLR signaling cascade [41; 42]). Lack of these proteins has also been associated with a functional reduction in the activity and nuclear translocation of the major pro-inflammatory NF-κB and p38/JNK transcription factors [41; 42]. However, most recent data suggest that the mechanisms responsible for the diminished cytokine response in preterm infants lie downstream of gestational changes in PRR expression ([32; 43] and below).

The fundamental mechanisms underlying the developmental attenuation of preterm innate immune cytokine responses have not been investigated. Epigenetic mechanisms have been largely implicated in the differentiation of hematopoietic cells [44]; therefore, it is plausible that similar mechanisms may play a role sustaining the developmental program regulating cytokine gene expression in early life. In newborns, lack of expression at the IL12A locus (encoding the p35 IL-12 subunit) in dendritic cells primarily occurs through nucleosome remodeling [45]. This lack of IL-12 expression is compensated for by a high production of IL-23 (through pairing of p19 with the p40 molecular subunit) in term neonates [46; 47; 48]. Recently, we have shown that preterm infants lack expression of p40 and therefore have a markedly reduced capacity to produce both IL-12 and IL-23 [34] (illustrated also in figure 1). In another recent study, reduced TLR3 expression in cord blood monocyte-derived dendritic cells was associated with allelic skewing of the TLR3 gene expression and modifications in chromatin structure, again suggesting a common role for epigenetic regulation of TLR function in early life [49]. However, whether and how epigenetic mechanisms regulate the maturation of innate immune functions during fetal life and in preterm neonates deserve more direct investigations.

3. Clinical impact of attenuated preterm innate immune functions

Fragile preterm infants are particularly susceptible to organ damage which may result from sepsis (reviewed in [50]). Infection can result in meningitis, which carries a major risk of permanent neurological impairment. Hemodynamic instability also carries a high risk of causing additional hypoxic-ischemic organ injury as it may occurs at critical developmental stages in preterm infants [51]. Given the unique vulnerability of preterm infants’ developing organs, it is likely that a reduced inflammatory response in fetal life serves to protect against the potential damaging effects of an excessive immune activation. However, the evolutionary advantage of attenuated innate immune defenses in utero clearly becomes a major clinical disadvantage following a preterm birth. Nonetheless, direct evidence for the implication of specific innate immune deficits in increasing preterm infants’ risk of infection in humans is lacking and inference from data in mice is complicated by significant functional differences across species [52]. Moreover, the presence of compensatory mechanisms (i.e. adaptive immune functions, etc.) at a later age may mask our ability to infer knowledge obtained from adults or from older children with primary immune deficiencies [53]. Despite these limitations some recent examples shed light on the clinical impact of specific immune deficits in human preterm infants. For example, the PRR Mannose-Binding Lectin (MBL) recognizes carbohydrate structures on the surface of a wide variety of pathogenic micro-organisms. The main function of MBL is to facilitate phagocytosis through antibody-mediated and C1-independent complement pathways [54]. In addition, MBL directly enhances TLR activity in the phagosome [55] and thereby can modulate inflammatory responses [56]. Reduced MBL serum levels have been linked to an increased risk of infection [57; 58; 59; 60; 61]. Preterm infants generally display lower MBL serum levels [58]. Moreover, common functional polymorphisms in the MBL2 gene resulting in variations in MBL serum levels may further increase susceptibility to infection in some infants [62; 63].

As mentioned above, preterm neonates who developed early-onset neonatal sepsis also display significantly lower serum levels of the IL-12/23 p40 protein subunit at birth compared to control neonates who did not go on to develop sepsis [34]. P40 mainly functions in conjunction with either p35 (to form the IL-12 cytokine) or p19 (to form IL-23), to support differentiation and maintenance of the major T helper 1 (Th1) or T helper 17 (Th17) cell regulatory subsets, respectively [64]. Data obtained from p40-deficient mice demonstrate a key role of these cytokines in protecting against mucosal pathogens [64]. Altogether, these data indicate an important role of specific innate immune defense pathways in protecting preterm infants against infection. However, data have largely been obtained from cord blood and a more detailed characterization of immunological deficits during the neonatal period, in humans is required in order to fully understand the factors responsible for their unique vulnerability to infection.

3.1 Pathogen-specific preterm innate immune responses

Although specific preterm innate immune deficits have been reported, little is known also about how these deficits interact while combating common neonatal pathogens such as Escherichia coli, Candida, Staphylococcal species or group B Streptococci (GBS). The lack of data stems, in part, from great ethical challenges with the conduct of studies requiring peripheral blood in vulnerable newborns. However, recent improved methods for polyfunctional assessment of immune functions from amounts of blood obtainable in small preterm neonates greatly facilitate investigations of pathogen-specific responses to common neonatal infections [32; 33; 43; 65; 66].

Coagulase-negative staphylococci (CoNS) species is one of the most common source of blood stream infection in the neonatal intensive care unit [33]. In mice, TLR2 appears to be particularly important in the early detection and clearance of Staphylococcus epidermis especially in conditions of reduced bacterial load [66]. TLR2 appears to play an important role in the innate immune recognition of this pathogen by human neonatal blood cells [66; 67]. On the other hand, adult- or term-like levels of TLR2 expression is preserved in preterm neonates, suggesting that their higher vulnerability to CoNS arise from more downstream functional differences [30; 41; 43].

The responses to GBS, another major neonatal pathogen, have also been recently examined more specifically in vitro [32]. Interestingly, the ability of preterm infants’ cord blood monocytes to phagocytose GBS is comparable to term neonates or adults [32]. However, the cytokine response produced upon exposure of preterm cord blood mononuclear cells to live or heat-killed GBS was significantly impaired, again suggesting downstream attenuation of the innate immune response to this pathogen [32]. Altogether, these data highlight the importance of a more thorough characterization of innate immune deficits in the context of neonatal pathogens to facilitate a targeted development of pharmacological strategies either to promote immune defenses or to limit invasive medical interventions at times when infants are most vulnerable immunologically.

4. Potential adverse effects of an excessive innate immune activation in preterm infants

Dysregulated innate immune responses play a major role in the etiology of common and serious preterm neonatal complications, such as bronchopulmonary dysplasia (a form of neonatal chronic lung disease) and necrotizing enterocolitis (a surgical intestinal complication) ([68; 69] for some recent studies). Apart from the direct effect of infection, most recent evidence indicate that non-infectious stimulation of the innate immune system may also present additional risks to preterm infants [70]. Supplemental oxygen, mechanical ventilation and intravenous nutrition are often used as life-supporting therapies in their initial weeks of life. Because of a close interplay between cellular oxidative stress and innate immune sensors these intensive care interventions also lend infants to experience significant inflammation resulting from an oxidative stress [70; 71; 72]. According to recent data in human infants, brief periods of oxygen administered at birth may generate sufficient inflammation and oxidative injury to cause sustained organ damage [71]. This oxidative stress is likely exacerbated by a profound immaturity of anti-oxidant defenses [73], which continues to drive a controversy in neonatal care about the amount of supplemental oxygen that is safe to administer to preterm newborns [74]. These data clearly mandate a proper understanding of the impact of therapeutically altering the normal preterm homeostatic innate immune balance in infants exposed to intensive care, before responses can be safely manipulated for the purpose of preventing infection.

5. Other emerging research areas

Other recent findings in neonatal immunology have expanded the scope of future translational investigations, while highlighting a number of important research questions. For example, in small series the intestinal microbial flora appeared distinct in preterm infants who develop sepsis, suggesting a potentially acquired predisposition [75]. Feeding human breast milk to premature infants reduces the incidence of necrotizing enterocolitis and favorably impacts their health in a number of other ways [76], whereas broad-spectrum antibiotic exposure was associated with a higher incidence of necrotizing enterocolitis [77; 78; 79]. Also, exogenous administration of certain types of bacteria through the administration of probiotics (e.g. Bifidobacteria and Lactobacillus) reduces the incidence of necrotizing enterocolitis in high-risk preterm infants [75; 80; 81; 82; 83]. In mice, exposure to breast milk immediately postpartum modulates TLR reactivity in the intestinal epithelium [84]. Altogether, these data highlight the potential therapeutic benefit of manipulating preterm infants’ own microbial flora in order to reduce serious complications such as infection and/or necrotizing enterocolitis. These data also raise more fundamental questions about the importance of the human microbiome in shaping the developmentally immature immune system.

Finally, some recent data indicate that early life exposure to infection may have life-long effects on the immune system [85; 86]. Based on animal studies, neonatal infections have significant long-term “programming” effects resulting in long-term inflammatory diseases [87; 88; 89; 90]. However, whether similar long-term effects occur in humans is completely unknown. Given their high rate of infection and their unique immunological vulnerability, the long-term immunological consequences of an early life exposure to infection in preterm infants certainly deserve more investigations.

6. Conclusions

In summary, a focused drive to improve knowledge of the human innate immune system at the very early stage in life is important in order to reduce neonatal mortality and achieve better health outcomes in preterm neonates. Major knowledge gaps remain to be filled in order to orient future clinical research, mainly in our understanding of the fundamental mechanisms regulating specific developmental innate immune pathways and how deficits in these pathways contribute to the overall risk of infection during the neonatal period and in the context of common neonatal infections. Finally, it is unclear how the preterm immune system can be safely modulated in order to avoid potential harmful consequence of an excessive immune activation on fragile immature organs. From a basic science perspective, a functional characterization of the immune system before the emergence of protective adaptive immunity also offers unique opportunities to understand the hierarchical importance of innate immune functions in humans.

Acknowledgments

The corresponding author’s (PML) research is funded by Hospital for Sick Children Foundation (XG09-015R) and Canadian Institutes of Health Research grants (MOP-110938). We thank Linda Dix-Cooper for an editorial revision of this manuscript. AAS is supported by a Child & Family Research Institute Graduate Studentship. PML is support by a Clinician-Scientist Award from the Child & Family Research Institute and a Career Investigator Award from the Michael Smith Foundation for Health Research.

Abbreviations

CoNS

Coagulase-negative staphylococcus

GBS

Group B streptococcus

MBL

Mannose-binding lectin

miRNA

regulatory micro-ribonucleic acids

NLR

Nucleotide Oligomerization Domain (NOD)-like receptors (NLR)

PRR

Pattern-Recognition Receptor

RLR

retinoic acid-inducible gene I (RIG-I)-like receptors

TLR

Toll-like receptor

Footnotes

1

Using keywords: [Human AND (immunity OR immunology)], [newborn OR neonate OR infant] and [premature OR preterm], respectively.

References