Inflammation and the Newborn Brain

Neutrophils

Neutrophils are polymorphonuclear immune cells derived from myeloid cell lineage. After an infection or tissue damage, they are the first immune cells recruited to the site of infection. As these cells are freely flowing in the circulation, they respond to cytokines and chemokines (e.g., tumor necrosis factor [TNF]-α; IL-1) or to microbial products (e.g., endotoxins) released at the infection site. They migrate along blood vessels through interactions of adhesion molecules on their surface with endothelial cells and reach the infected site by diapedesis. When activated, neutrophils form pseudopodia around microorganisms to phagocytize them.

Before 31 WG in humans, fetal neutrophil levels are low, and their chemotactic ability is limited. This limitation is due to lower amounts of adhesion molecules expressed on their surface compared with neonatal and adult neutrophils, thereby reducing their ability to adhere and extravasate from the circulation.

Depending on the environmental signals they receive, neutrophils can respond as proinflammatory or antiinflammatory agents or can participate in the growth and proliferation of other immune components. Each response is characterized by the secretion of a special repertoire of cytokines required for the roles of neutrophils and their crosstalk with other immune cells. For example, neutrophils are crucial mediators of the T helper 17 (Th17) pathway of resistance to pathogens. Cytokines derived from Th17 (IL-17, IL-8, interferon [IFN]-γ, TNF) favor the recruitment, activation, and prolonged survival of neutrophils at inflammatory sites.

Another feature of neutrophils is their capacity to form extracellular fibrillary networks, known as neutrophil extracellular traps. These networks are composed mainly of DNA and proteins from neutrophil granules, such as myeloperoxidase and neutrophil elastase, and can trap microorganisms.

Monocytes

Monocytes derive from the myeloid cell lineage and belong to the mononuclear phagocyte system. They reside in the circulation before their recruitment to the tissues, where they differentiate into macrophages. Of note, macrophages are abundant in the meninges, choroid plexus, and in the vicinity of brain vessels in close contact with endothelial cells (perivascular macrophages). Monocytes can influence the adaptive immune response through cytokine secretion and antigen presentation. Under inflammatory conditions, they can differentiate into conventional dendritic cells (DCs).

Sixty days after conception, hematopoiesis is established in the fetal liver, and monocytes are present in high amounts in the circulation. Their counts, phagocytic function, and antigen-presenting capacity increase with gestational age.

Microglia

Microglia are the macrophages of the brain and the predominant cells of the immune responses of the CNS. Under physiological conditions, they sense the brain environment to regulate homeostasis (e.g., neuromodulation, synaptic homeostasis and plasticity, phagocytosis of apoptotic neurons and debris). In adults, microglia tile the entire brain volume via ramified processes, and these cells specialize in surveying the parenchyma. Their sensing functions are largely linked to a plethora of microglial receptors, including receptors of cytokines/chemokines and damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs), for the detection of changes in the brain environment. A relevant example of a DAMP is a specific molecular motif of the lipopolysaccharide (LPS) of gram-negative organisms, which activates toll-like receptor (TLR)-4 and cluster of differentiation (CD)14 on brain microglia, resulting ultimately in generation of reactive oxygen species (ROS) and other molecules that lead to the death of preoligodendrocytes (pre-OLs) (see Chapter 19 ). An example of a PAMP is hyaluronic acid (HA), generated by the reactive component of the diffuse astrocytic gliosis of preterm cerebral white matter injury (see Chapter 18 ); arrest of pre-OL maturation is stimulated both in vitro and in vivo by high molecular forms of HA that are digested to bioactive forms by hyaluronidases in the extracellular matrix (see Chapter 19 ).

CNS diseases or injury, and any other type of disturbance to the CNS, may trigger the conversion of homeostatic microglia into “reactive” or “activated” microglia. Activated microglia have been shown to adopt a considerable variety of immune phenotypes in the brain. The classical M1 versus M2 nomenclature used to refer to the inflammatory versus proresolution/alternatively activated microglial phenotypes has been the subject of considerable debate. Microglial activation is a dynamic response that is regulated both temporally and spatially, depending on the specific nature of the brain injury. Microglial density and activation status are also modulated by the microenvironment of brain regions. However, three main phenotypes can be distinguished: (1) a proinflammatory phenotype, with high levels of expression of messenger RNA encoding several proinflammatory enzymes or cytokines used as markers, such as NO synthase inducible 2, cyclooxygenase 2, and TNF-α; (2) an antiinflammatory and proregeneration phenotype, with the overexpression of markers such as arginase 1, CD-206, lectin galactosidase-binding soluble 3, and insulin-like growth factor 1 (IGF-1); and (3) an immunoregulatory phenotype often associated with proinflammatory cytokines, probably to prevent microglial overactivation and to resolve inflammation, with the expression of various markers, including IL-1 receptor antagonist (IL-1RA), suppressor of cytokines signaling 3, and IL-4 receptor α. These phenotypes may overlap, and transitions between them have been observed, at least in vitro.

The stratification of microglial cell populations has been considerably refined thanks to the emergence of technologies for genome-wide expression profiling at the single-cell level. The common patterns that have emerged from single-cell RNA sequencing (RNAseq) studies have provided new insight into the delineation of subtypes of microglial cells, particularly for those associated with disease and inflammation. The first new microglial subtype described on the basis of single-cell RNAseq approaches was disease-associated microglia in mouse models of Alzheimer disease and amyotrophic lateral sclerosis. In the other neuroinflammatory or pathological conditions tested, complex patterns of microglial heterogeneity were observed, with disease/condition-specific microglial activation states displaying low levels of expression of homeostatic microglial marker genes, such as P2ry12, Siglech, Sall1 Cx3cr1, and Tmem119.

The approach of defining single cell transcriptomics has led to the discovery of many distinct microglial subpopulations with unique molecular signatures. These distinct subpopulations have been known to change over the course of development of mouse brain and to exhibit regional specificity. At least nine distinct microglial subpopulations have been defined. Notably a distinct microglial phenotype has been localized to developing white matter tracts. This subpopulation, termed axon-tract associated microglia, has molecular signatures consistent with involvement in axonal development just before myelination (i.e., the time period occurring in the premature brain). It could be speculated that, if activated to a proinflammatory state, this subpopulation could contribute importantly to white matter injury and dysmaturation, as described in Chapters 18 and 19 .

In neonatal brain injuries, the developmental dynamics of microglia are important to consider. The populations of microglia present in the prenatal and early postnatal brain have a specific identity and morphology; they generally have no ramifications and are ameboid in shape. Microglial mesoderm progenitors arise from the yolk sac (79, 80) and penetrate the human brain at 4.5 to 5.5 WG via the choroid plexus, meninges, and ventricles, at developmental stages matching those at which this event occurs in animal models, including rats (embryonic day [E]11) and mice (E8). These ameboid and proliferating microglia form clusters during development (1) between the subplate and cortical plate (10 to 12 WG) where the first synapses are detected; (2) in the corpus callosum (16 WG); and (3) around the anterior horn of the lateral ventricle, the site of major axonal crossroads (19 to 30 WG). This accumulation of ameboid and proliferating microglia at the site of white matter injury in premature newborns most likely contributes to the vulnerability of the white matter in these infants.

Natural Killer Cells

NK cells are granular immune cells that belong to the lymphoid cell lineage and play essential roles in immunity against viruses and the immune surveillance of tumors. They are a potent innate source of IFN-γ. Their immunoglobulin (Ig) receptors bind antibody-coated targets leading to antibody-dependent cellular cytotoxicity. They also recognize abnormal cells lacking major histocompatibility complex (MHC) class I and receive activation signals leading to the secretion of perforins onto the surface of target cells. The formed pores allow NK-derived granzymes to enter the cytoplasm and induce the apoptosis of the target cells.

NK cells are first detectable in the fetal liver at 6 WG, and their levels reach a maximum at birth. Although they are part of the innate immune system, recent studies have proved that NK cells have some traits of the adaptive immune system. For instance, they sometimes rely on interactions with myeloid lineage cells for potent effector function (a phenomenon known as priming). Furthermore, some previously activated NK cells show immunological memory, resulting in their performing as better effectors upon rechallenge.

Dendritic Cells

As part of the innate immune system, DCs modulate the adaptive immune response and immune tolerance. These phagocytic cells express several innate pattern‑recognition receptors (PRRs) that bind pathogen-associated molecules, resulting in increased mobility for migration. DCs are the main antigen presenting cells (APC), and they determine the fate and functionality of antigen-specific T-cell responses. When activated, DCs produce cytokines and express costimulatory molecules necessary for priming of naïve T cells and stimulation of effector T cells.