Impact and Clinical Implications of Prematurity on Adaptive Immune Development

Abstract

Purpose of Review:

Detail normal adaptive immune maturation during fetal and neonatal life and review the clinical implications of arrested immune development.

Recent Findings:

Advancements in the field of immunology have enabled investigations of the adaptive immunity starting during fetal life. New insights have drawn important distinctions between the neonatal and adult immune systems. The presence of diverse immunologic responses in the perinatal period suggests the importance of in utero immune development. Disruption of immune maturation due to premature birth may have significant implications for clinical pathology.

Summary:

Establishing protective adaptive immunity during the perinatal period is critical for effective immune responses later in life. Preterm infants are susceptible to aberrant immune system maturation and inflammatory immune responses have been associated with the development of necrotizing enterocolitis (NEC) and bronchopulmonary dysplasia (BPD). Improving our understanding of how immune responses contribute to the pathogenesis of NEC and BPD may offer new opportunities for future treatment and prevention of these diseases.

Introduction:

Throughout human life individuals are exposed to countless antigens, both innocuous and pathogenic, and it is the immune system’s responsibility to respond appropriately to these encounters. Analogous to our sensory system, the immune system reacts to external stimuli that contribute to its activation and development. This process begins during the first trimester and continues throughout life. Infants are more susceptible to infectious diseases than adults, with prematurity conferring additional risk, suggesting differences in protective immunity exist with age (1, 2). Preterm infants are also affected by inflammatory diseases that have a basis in immune dysregulation, particularly necrotizing enterocolitis (NEC) and bronchopulmonary dysplasia (BPD) (3, 4). The perinatal period is a critical time point for the development of protective immune responses that establish a foundation for maintaining immune homeostasis and disease prevention throughout life (5).

Our understanding of the processes underlying early life immune development have recently improved resulting in the recognition that the infant immune system is distinct from adults (1). As early life immune responses are highly influenced by the conditions in which they are formed, a deeper understanding of how preterm birth impacts immune development may elucidate mechanisms through which NEC and BPD arise and augment our ability to treat and prevent these disease entities. We will first review what is currently known about normal immune development, with a special focus on the T cell compartment of adaptive immunity, and how preterm birth affects healthy development. Lastly we will examine the immunopathology of NEC and BPD.

Briefing on immunity:

The immune system is comprised of the innate and adaptive limbs, two separate branches that have distinct roles and work in coordination to promote self-tolerance while providing protection from pathogens. The innate immune system, made up in part by macrophages, neutrophils, and dendritic cells (DC), is capable of rapid responses but does not generate memory to previously encountered pathogens. The adaptive immune system, composed of T and B lymphocytes, is capable of recall following initial pathogen encounters allowing faster and tailored responses during repeat exposures (Table 1). CD8⁺ T cells control cell mediated immunity by governing the clearance of intracellular pathogens through the release of cytotoxic mediators. CD4⁺ T cells coordinate multiple aspects of the immune response by the secretion of a complex set of cytokines and chemokines enabling the recruitment of other immune cell subsets, and facilitating antibody production by B cells. B cells primarily produce and secrete protective antibodies, but can also serve a number of other functions including cytokine production and regulation of wound healing (6). Conventional T cells express a T cell receptor made up of α and β chains (αβ T cells) while a smaller proportion of T cells express g and d chains in their TCR (γδ T cells). The majority of this review will focus on conventional αβ T cells, however it is important to note that γδ T cells are found in their highest proportions in the intraepithelial layer of the intestines, where they play a key role in maintaining mucosal health and protection (7). For more in depth reviews on adaptive immunity please see references (8–10). Optimal immune protection depends on the production of neutralizing antibodies by memory B cells along with the generation of antigen and site specific T cell memory.

Table 1.

Categorization and functional description of key adaptive immune cell subsets.

Subset	Role in Immune Response	Effector Function
CD4+ T cell	Coordinate immune responses and facilitate antibody production by B cells through the release of cytokines and chemokines	Dependent on subset and location
CD8+ T cell	Control cell mediated immunity by governing the clearance of intracellular pathogens through the release of cytotoxic mediators	Granzymes, Perforin
B cell	Produce and secrete protective antibodies; produce cytokines; regulate wound healing	Antibodies
TRM	CD4+ or CD8+ memory T cell, provide protection and maintain mucosal homeostasis in tissue of prior antigenic exposure	Dependent on specificity and location
Th1 cell	CD4+ memory T cell mediates eradication of intracellular pathogens through activation of macrophages and CD8+ T cells.	IFN-γ, TNF-β, IL-2
Th2 cell	CD4+ memory T cell which coordinate clearance of extracellular pathogens by activation of eosinophils, mast cells. Stimulate IgE production from B cells.	IL-4, IL-5, IL-10, IL-13
Treg	CD4+ T cells responsible for maintainance of self-tolerance, limiting inflammation and immune homeostasis.	IL-10, IL-33, TGF-β, Amphiregulin
Th17 cell	CD4+ memory T cell with role in mucosal homeostasis, pathogen clearance, and regulation of neutrophils.	IL-17A, IL-22, TNF-α

The T cell compartment of the adaptive immune system is capable of differentiating into a vast array of memory subsets that coordinate immune responses and mediate protection. Memory CD4⁺ T cells, which are also known as helper T (Th) cells, can be divided into several major subtypes including: Th1, Th2, Th17, and T regulatory cells (Tregs) (Table 1). The balance between Th1 and Th2 cells is particularly important for effective immune responses (11). In general, Th1 cells play a role in macrophage activation and cytotoxic eradication of intracellular pathogens. Th1 cells produce the inflammatory cytokines interferon (IFN)-γ, tumor necrosis factor (TNF)-β, and interleukin (IL)-2, key for anti-microbial eradication and T cell differentiation (11). Th2 cells are primarily responsible for extracellular pathogen clearance. Th2 cells initiate immune responses from eosinophils, mast cells, and B cells by the secretion of IL-4, IL-5, IL-10, and IL-13 during effector responses. Importantly, as B cells increase production of IgE following stimulation by Th2 cytokines, over stimulation can contribute to allergic reactions through hyper-responsiveness to innocuous antigens (11–13). Tregs play an essential role in maintaining self-tolerance, immune homeostasis, and limiting inflammation by controlling the activation, proliferation and cytokine production of other CD4⁺ and CD8⁺ T cell subsets (14, 15). Interestingly, Tregs have been recently found to play an important role in tissue repair following injury (16). Lastly, Th17 cells function to control extracellular pathogens and fungi by producing the pro-inflammatory cytokines TNF-α and IL-17. Importantly, these cytokines are known to impact the growth and development of cells in many tissues (17). Ultimately the establishment of memory subsets with distinct and varied functionality contributes to a lifetime of pathogenic or protective immune responses.

Memory T cells can exist as both circulating and non-circulating subsets. Circulating memory cells have the ability to move throughout the blood, enter lymph nodes, and survey mucosal sites. Non-circulating memory cells, often referred to as tissue resident memory T (T_RM) cells, are a specialized subset that provide protection and maintain mucosal homeostasis in the tissue where they’ve previously encountered antigen (Table 1). Importantly, the conditions present during memory T cell establishment can result in skewed and allergic future responses, highlighting the influential role of environmental factors (18). Additionally, T_RM cell dysregulation is associated with mucosal inflammation and autoimmune diseases, contributing to lifelong chronic illnesses (19). As early life immune responses are formative and can have profound implications for health, it is important to understand the role of disrupted immune memory development in the pathogenesis of diseases in the neonatal setting.

Early Life Immunity

Neonatal life is a unique developmental time period that is marked by a remarkably large influx of novel antigens and a high level of plasticity. Neonatal immunity has historically been defined as immunodeficient, a theory originating from reductions in proliferation, capacity, and IL-2 production (20). However, pivotal studies conducted in the 1990s revealed the neonate’s ability to produce mature adult-like responses under the proper conditions, redefining early life immunity as distinct from adults rather than immunodeficient (21–23). Previously, much of what we understood about early life immunity was derived from mouse models and human cord blood, which may not accurately represent human neonatal immunity (24). Recent advancements in blood collection and innovative methods to study immune responses within mucosal and lymphoid tissue have significantly improved our ability to study and track the development of the immune system in early life, revealing the importance of proper immune development during this critical period of life (25–28).

Recent studies demonstrate that the adaptive immune system begins developing as early as the end of the 1^st trimester of gestation and continues to mature and accumulate phenotypic diversity through birth (1, 28, 29). Importantly, profound changes in the immune systems composition occurs shortly after birth, influenced by the multitude of new pathogen exposures. Olin et al showed that infant’s immune profiles, including B cells, natural killer cells, and DCs, follow a common pattern of compositional changes and converge to become representative of adult-like phenotypes by 3 months (24). This convergence is dependent on postnatal pathogen exposures which influence the acquisition of immune variations observed in healthy adults. Thus, the neonatal period is crucial for the normal development of the immune system and deviations may have a profound impact on future health.

Early Life T Cell Composition

The perinatal immune system must mediate protection from harmful pathogens while also maintaining self-tolerance and avoiding excessive activation. Newborns transition from the protection of the womb to an environment with a myriad of pathogenic and innocuous antigens, and maintain a distinctly tailored T cell configuration. Early life tolerance is thought to be achieved through a skewing towards Th2 responses and a prominent role for Tregs. Tregs can be detected as early as the 2^nd trimester and increased proportions of Tregs can be found throughout all tissues during the first few years of life compared to adults(25, 30). Evidence suggests that naïve neonatal T cells possess an inherent bias to develop into Tregs and early life Tregs exhibit heightened suppressive capabilities, implying an innate tendency towards tolerogenic responses (25, 31). Infant immune responses additionally control inflammation by limiting the production of pro-inflammatory cytokines (32–34). A bias towards Th2 responses in early life is not fully understood, however evidence for several mechanisms exist; 1) Increased proportion of Th2 cells during fetal life, which persist postnatally, 2) following antigenic re-exposure early life Th1 cells are more susceptible to undergoing apoptosis than Th2 cells and 3) epigenetic modifications favoring Th2 differentiation (35). Overall, the functional and compositional variability observed in the early life T cell compartment is distinctly structured towards anti-inflammatory and tolerogenic responses.

Memory Formation and Compartmentalization

Much of the emerging evidence surrounding adaptive immunity has shown that localized tissue specific responses are key for optimal protection from pathogens. Tissue-specific compartmentalization allows memory cells to respond at the site of previous antigenic exposure while harboring functional capacities specific to the needs of the local tissue. Compartmentalization of T cell regulation and differentiation is established in mucosal and peripheral tissues during infancy and continues to develop throughout life (36). There exist quantitatively less memory cells in pediatric mucosal and lymphoid tissues compared to adults, suggesting accumulation occurs over the course of life (25). Importantly, emerging evidence points to the intestinal mucosa as a key site for early memory formation.

Beginning as early as 16 weeks of gestation, the fetus has already established remarkably complex mucosal memory compromised of mature and phenotypically distinct subsets (26, 27). Studies indicate that memory accumulation in the fetal intestine is directly correlated with gestational age with higher proportions of memory T cells found closer to term (27). Moreover, fetal intestinal memory T cells demonstrate the phenotypic and transcriptional signature of resident memory (26, 27). The specificity of these memory T cells is not yet defined, however, the presence of other maternal antigens and bacterial byproducts in the amniotic fluid, may be responsible for prompting antigen-driven memory development in fetal intestines (37). As T_RM cells are essential for optimal protective responses, fetal memory formation suggests the importance of an in utero developmental period which may provide a vital foundation for protective responses during infancy.

Despite the evidence for early development of protective memory subsets, infants are more susceptible to infectious diseases compared to older children and adults (2). Both mouse and human studies have revealed that T cells in early life are transcriptionally programmed to rapidly proliferate into cells which generate strong effector responses at the expense of T_RM formation (38–40). Addtionally, effector and memory T cell responses are primarily confined to the mucosal sites of the lungs and small intestine in infants with significant accumulation in lymphoid tissue occurring much later in life (25). Importantly, the rapid accumulation of effector responses in mucoscal tissue has been associated with disease severity during viral respiratory tract infections in infants (40, 41). Taken together, infants appear adept at generating strong adaptive immune responses capable of eradicating harmful pathogens but these adaptations may contribute to immunopathology and decreased future protection. Fostering the conditions that promote protection over pathology may prevent acute pathology and improve lifelong mucosal health.

Fetal Intestinal Inflammatory Potential

The acquisition of immunological memory early in utero serves to facilitate intestinal growth and development but is capable of inducing an inflammatory environment. TNF-α producing CD4⁺ T cells are detectable from the beginning of the first trimester (28). Schreurs et al showed that TNF-α producing CD4⁺ T cells have a dose dependent effect on intestinal epithelial growth, with low levels supporting development and high levels impeding growth (28). T cells secreting IFN-γ IL-2, IL-4, granzyme B, and IL-17A have been identified within the developing intestine, suggesting additional mechanisms of inflammation within the fetal intestine mediated by the adaptive immune system(26). Altogether, the evidence supports that a delicate balance, requiring strict control, exists during the development of the intestinal mucosa and that disruptions within this balance can have profound deleterious effects.

Effect of Premature Birth on Immune Development

Significant improvements have occurred in the survival rate of preterm infants, however, diseases associated with preterm birth, such as NEC and BPD, remain a persistent cause of morbidity and mortality in preterm infants (42, 43). Infants born prematurely harbor immunologic profiles akin to fetal immune composition and functionality, and therefore may not be as well-equipped as full term infants for the ex utero environment. Preterm infants overexpress genes responsible for the negative regulation of IFN-γ production, T cell proliferation, and IL-10 secretion (24). Extremely preterm infants have been shown to possess hyperresponsive T cells with decreased inhibitory molecules (44). Addtionally, infants born prematurely harbor increased levels of naïve CD4 T cells which, when failing to transition to a “full term” phenotype, result in increased respiratory morbidity (45). Importantly, the differences in immune profiles of preterm and term infants have been noted to become even more dissimilar at 3 months of age suggesting that with maturity, immune discrepancies are not alleviated but are further exacerbated (24). The arrested development of immune maturation due to prematurity leads to structural, compositional, and functional immunologic abnormalities with potentially lifelong implications.

Adaptive Immume System Immunopathology of Necrotizing Enterocolitis

NEC is a severe inflammatory disease driven by mucosal dysbiosis resulting in disruption of the intestinal epithelium, which can lead to long-term, debilitating gastrointestinal and neurocognitive outcomes (3, 46–48). The intestine is a highly immunoreactive tissue and during NEC cascades of pro-inflammatory signaling including cytokines, immunoglobulins, and immune cells are associated with significant tissue damage (for reviews on NEC, see refs. (42, 49)). Pathways of immune dysregulation in NEC may include the imbalance of CD4⁺ effector T cells, γδ T cells, Th17, and Treg subsets and their associated cytokines (Fig. 1). Elucidating dysregulated adaptive immune responses that drive morbidity during NEC will help to define mechanisms by which disease prevention and intervention can be targeted.

Figure 1: Adaptive Immune Changes During Necrotizing Enterocolitis.

Left; Healthy intestine with γδ T cells populating the intraepithelial layer, regulatory T cells (Tregs) secreting IL-10 to limit inflammation, and memory CD4 T cells producing low levels of TNF-α to promote mucosal development. Right: Necrotizing Enterocolitis affected tissue with loss of γδ and TReg subsets, increased presence of Th17 subsets and increased production of TNF-α by CD4 memory T cells.

Mouse models and neonatal tissue sampling have shown that the intestinal immune composition during NEC is distinct from that of healthy tissue with several potentially targetable pathways identified. Tissues obtained from infants with NEC have been found to have increased levels of of TNF-α producing CD4⁺ effector T cells with upregulation in genes for TNF-α signaling (28). Interestingly, the role of IL-17 in mediating protection or pathology may depend on the T cell subset responsible for its secretion. IL-17 production by intraepithelial γδ T cells contributes to intestinal barrier protection and prevention of bacterial translocation (50). The recruitment of IL-17 secreting CD4⁺ T cells via a TLR4 dependent pathway, causes apoptosis and reduced enterocyte proliferation ultimately resulting in NEC (51). Immunoregulation and homeostasis maintenance through IL-10 is important for NEC prevention, with decreased levels of IL-10 associated with the development of NEC (Fig. 1) (52, 53). Tregs are a major source of IL-10 and a decreased proporition of Tregs in the lamina propria has been found in infants with NEC (54). Evidence from experimental models indicates that maternal breast milk increases production of intestinal IL-10. Additionally, the maternal milk of infants who develop NEC contains less IL-10 (55, 56). Prematurity results in many immunologic variations that can trigger the disruption of intestinal homeostasis providing a potential explanation for why NEC is a rare pathology in full-term infants (57). A deeper understanding of the impact of arrested mucosal immunity development may allow for the introduction of effective therapies to promote proper immune responses and improve outcomes related to NEC in preterm infants.

Immunopathology of Bronchopulmonary Dysplasia

BPD is a complex, multifactorial disease caused by disrupted lung development leading to significant morbidity and mortality in low birth weight preterm infants (for reviews on BPD, see refs. (58–60)). While no single mechanism underlying disease progression has been described, multiple intrinsic and external factors have been identified in the pathogenesis of BPD (61). Identifying targetable immunologic pathways and the modifiable environmental factors resulting in excessive immune activation may be key to prevention and improving outcomes. Pathways resulting in inflammation and hyperresponsiveness are central to our current understanding of BPD. Ambalavanan et al demonstrated that during the first 28 days of life serum concentrations of specific cytokines are correlated with the development of BPD. Higher levels of IL-1β IL-6, IL-8, IL-10, IFN-γ, and lower levels of IL-17, RANTES, and TNF-β were observed to be associated with concomitant development of BPD (62). The inherent Th2 bias of early life may also contribute to BPD pathogenesis. The presence of chemokines, eotaxin-2 and MCP-4, which are induced by Th2 responses have been linked to the development of BPD (4). Eotaxin-2 and MCP-4 induce the recruitment and activation of eosinophils which can ultimately result in allergic responses and eosinophila has independently been associated with the development of BPD in several studies (63, 64). Whether these pathways are induced by modifiable factors which could represent opportunities for intervention remain to be determined.

One modifiable factor that has been linked to immune activation is the administration of excessive oxygen. Hyperoxia in a mouse model of BPD resulted in significant hypermethylation of the PI3K-AKT pathway. This pathway ultimately controls key aspects of the adaptive immune system, particularly the differentiation of B and T cells (65). Hyperoxia can also induce hypermethylation of the TGF-β pathway resulting in the stimulation of apoptosis (66). Additionally, hyperoxia during the neonatal period is associated with suppression of genes involved in B and T cell activation with changes persisting up to 3 months after exposure (67). Alterations in these pathways may have a direct effect on protective immune responses, resulting in decreased recruitment of B and T cells to the pulmonary environment during viral respiratory tract infections leading to a lasting impact on future immunity. Continued optimization of non-invasive ventilation and vigilance with oxygen therapy should be encouraged as they represent opportunities to impact aberrant immune activation with implications for acute and long-term outcomes in BPD.

CONCLUSIONS

Emerging evidence and advancements in our knowledge of early life immunity have allowed us to recognize the distinctions of perinatal immunity that render its capabilities unique to the developing fetus and infant. We now appreciate how early immune development begins, the delicate balance that exists between inflammation and tolerance, and understand how protective memory is formed in response to early life antigenic exposures. Prematurity results in arrested development of the immune system and increases the susceptibility of developing NEC and BPD. Future research to better understand the connections between immunity and the development of NEC and BPD is warranted. Studies investigating the modifiable factors contributing to disease pathogenesis, particularly those focused on site specific immunity, could inform treatment plans for preterm infants and significantly improve outcomes related to prematurity.