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Age-appropriate biology of neonatal neutrophils
Introduction
Unraveling the complexities of intrinsic mechanisms behind the normal transition of the suppressed in utero neutrophil into the fully operational postpartum cell has been a goal of neutrophil biologists for nearly a century. Even though neonatal neutrophils have been historically described as functionally deficient when compared with adult cells, these phenotypic and functional disparities have been evolutionarily perfected over centuries and are essential to the divergent environments and pathogenic challenges to which they are exposed. Compared to the oxygen concentration of 21% in the earth’s atmosphere, the intrauterine environment is exceedingly hypoxic, with measured oxygen concentrations in the range of 3% to 5%. Because of this low oxygen content, cellular suppressive mechanisms that stabilize or counter hypoxia-inducible factor-1α (HIF-1α) are vital to prevent the expression HIF-1α proinflammatory genes. After parturition and in the immediate postnatal period, the neonate will also be newly exposed to trillions of microbes that will become essential constituents of their microbiome and for which immune tolerance is essential to prevent an acute and robust inflammatory response.
Like other major organs, the immune system matures during fetal development with well-described cellular transformations originating from the earliest hematopoietic precursors in the yolk sac and progressing to the well-developed myeloid progenitor cells in the bone marrow. The survival of extremely preterm infants exposes the severity of these immaturity-related impairments that places them at high risk for infection and sepsis-related mortality. In this chapter we explore differences between neonatal and adult neutrophils, describe neutrophil maturation throughout pregnancy, and highlight variances in neonatal neutrophil proficiency by gestational age and how these differences may impact long-term outcomes following infection or sepsis.
Development
Fetal hematopoiesis originates in the extraembryonic yolk sac around the third week of embryogenesis, where a transient population of primeval myeloid, megakaryocyte, and erythroid cells is formed. , Around weeks 7 to 8 of gestation, however, genuine, self-renewing hematopoietic stem cells (HSCs) are derived from specialized intraembryonic endothelial cells located in the ventral wall of the descending aorta. These primitive HSCs will seed the thymus, liver, and spleen, where hematopoiesis will continue until the seventh month of gestation. , After this time, the bone marrow will become the primary source of red cells, white cells, and platelets. ,
Primitive neutrophil precursors initially appear in the human clavicular marrow between 10 and 11 weeks of fetal development and are detected in the peripheral vasculature by the end of the first trimester. , Mature neutrophils can be identified by 14 to 16 weeks postconception and are formed by HSCs positioned in specialized niches in the trabecular regions near the endosteum of the long bones, where they reside near osteoblasts. Once formed, neutrophils will exit the bone marrow into the bloodstream by traversing through the cell bodies of the bone marrow endothelium rather than through the cell junctions, by a process known as transcellular migration. ,
Neutrophils reside in three distinct groups, or pools, known as the proliferative (bone marrow storage and release), circulating (bloodstream), and marginating pools. These pools are maintained by a delicate physiologic balance that is closely regulated by the individual’s state of health and the maturational stage of the cell. Neutrophil homeostasis is also directly regulated by the controlled production of granulocyte–colony-stimulating factor (G-CSF), granulocyte macrophage–CSF (GM-CSF), interleukin-19 (IL-19), CXCL1, CCL2, and CXCL10 by nearby conventional dendritic cells. ,
Within the bone marrow, neutrophil development is defined by granule formation within the maturing cell in a process known as neutrophil granulopoiesis. Neutrophil granulopoiesis and maturation are stimulated by IL-19 produced predominantly by osteoclasts, which activates IL-19 receptor (IL-20Rβ)/Stat3 signaling in neutrophil progenitors to promote their expansion and facilitate their differentiation. Beginning between the myelocyte and promyelocyte stages of development, granulopoiesis proceeds over the subsequent 4 to 6 days to yield mature, segmented neutrophils ( Fig. 4.1 ). The formation of neutrophil granules is a continuous process by which granule proteins are packaged as they are produced in the process called “targeting by timing.” In general, azurophilic granules are synthesized in promyelocytes, specific granule proteins in myelocytes, and gelatinase granule proteins in metamyelocytes and band cells, after which granule formation concludes and secretory vesicles form. , , Because the formation of neutrophil granules occurs in a continuum, granule proteins may be found in more than one granule type. Direct sorting, shuttling, and packaging of granule proteins are important for proper neutrophil granule formation, with adaptor protein complexes (APs) and the monomeric Golgi-localized γ-adaptin ear homology ARF (GGA) binding protein playing key roles in this process. ,
Exocytosis of neutrophil granules occurs in the reverse order of their formation and in a hierarchic fashion based on the magnitude of the stimulus and the function of their contents (see Fig. 4.1 ). , Thus, minimal stimulation will trigger the extrusion of secretory vesicles first. Although secretory vesicles are not considered genuine neutrophil granules, they are important reservoirs of membrane-associated receptors that allow the neutrophil to establish firm contact with activated vascular endothelium and transmigrate into inflamed tissues. Next to degranulate are gelatinase granules, containing matrix-degrading enzymes (gelatinases) and membrane receptors, that are necessary for extravasation into inflamed tissues during early inflammatory processes. Specific granules follow and contain potent antimicrobial substances, which can be extruded either into phagosomes, that provide lethal enclosures for intracellular pathogen killing and clearance, or extracellularly. Last to degranulate are azurophilic granules, which are unique in that they are tightly controlled and can only be activated by potent stimuli. Once mobilized, these granules extrude highly toxic acidic hydrolases and microbicidal proteins into phagolysosomes. These structures provide specialized intracellular containment vacuoles that protect the surrounding cells and tissues from injury, which would occur if these noxious substances were to be expelled extracellularly.
Proliferative neutrophil pool
A delicate balance between granulopoiesis, bone marrow storage and release, intravascular margination, and transmigration into peripheral tissues is required to maintain neutrophil homeostasis. , Mitotic neutrophil precursors that comprise the proliferative pool are important in maintaining and replenishing neutrophil numbers, including myeloblasts, promyelocytes, and myelocytes. , In steady-state conditions, human adults produce nearly 100 billion neutrophils every day. These cells , are generally short-lived cells with a half-life of ~19 hours. In contrast, term neonates have greatly diminished neutrophil proliferative pools, estimated to contain only 10% adult values. While adults produce between 4 and 5 ×10 9 neutrophils/kg body weight per day, term newborns generate nearly a quarter and preterm infants only 20% of adult values. Moreover, the proliferative pool in a developing fetus or postpartum neonate experiences significant cell turnover, with more than two thirds of these cells residing in an active cell cycle. ,
Proliferative pool and the microbiome
The coevolution of mammals and their microbiota has led to direct regulation of the host neutrophil proliferative pool by microbial-derived mediators. In steady-state granulopoiesis, tonic microbial signaling leads to activation of the host systemic innate immune system via Toll-like receptor 4 (TLR4), a member of the highly conserved family of pattern recognition receptors that recognizes and binds to gram-negative lipopolysaccharide, pathogen-associated molecular patterns (PAMPs), and endogenous molecules generated as a result of tissue injury. This relationship is well demonstrated in adult mice, where the complexity of the intestinal microbiome directly controls the size of bone marrow neutrophil mitotic progenitors that function to maintain vigilance against potential pathogens.
In contrast to adults, the immune naïve fetus represents a unique challenge. In the course of natural parturition, newborns are exposed to and colonized by a vast number of microorganisms that comprise the maternal vaginal and rectal microbiota. Because these microbes harbor a variety of foreign nucleic acids, proteins, and antigens, neutrophil tolerance is imperative during the transition to extrauterine life to prevent robust systemic proinflammatory reactions during the establishment of the newborn’s microbiota. Interventions that impede the natural development of the newborn’s microbiota, such as cesarean delivery or exposure to intrapartum and/or postpartum antibiotics, may place the infant at an elevated risk for necrotizing enterocolitis, late-onset sepsis, prolonged length of stay, or even death.
To further explore the association of the microbiome and neutrophils in neonates, Deshmukh et al. used a murine model to demonstrate that induction of IL-17 by the pup’s intestinal microbiota resulted in activation of intestinal group 3 innate lymphoid cells. These lymphoid cells subsequently increased their production of G-CSF to enhance the proliferation of neutrophil mitotic precursors in a TLR4- and MyD88-dependent manner. This continuous, very low-level TLR signaling (below the threshold required for induction of adaptive immune responses) by microbial antigens and TLR ligands facilitates neutrophil homeostasis under steady-state conditions. Conversely, invading pathogens elicit an upregulation of TLR induction through detection of PAMPs, causing a rise of proinflammatory cytokines, including IL-1β, tumor necrosis factor (TNF)-α, GM-CSF, and G-CSF. , This surge of inflammatory mediators triggers emergency granulopoiesis, leading to an acute and drastic increase in the number of bloodstream neutrophils that are available to combat the ensuing infection. This acute proliferation of neutrophils can be attenuated in germ-free mice, where studies have shown severely delayed pathogen clearance and neutrophil recruitment following a challenge with apathogenic bacteria. Therefore maintenance of neutrophil homeostasis, proper cell functioning, and robust neutrophil proliferative during emergency granulopoiesis are all directly dependent upon the establishment of a healthy and robust microbiome after birth .
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