Migration and Adherence

Once released from the bone marrow storage pool, neutrophils are capable of migrating from the circulatory system into extravascular sites of inflammation. This process involves multiple steps, all of which show altered ability within the neonate. Following entry into the bloodstream, adherence to the vascular endothelial cells with subsequent leukocyte rolling and adherence is facilitated by cellular membrane molecules. As neutrophils transiently interact and roll along vascular cell surfaces they adhere to sites of inflammation through an interaction between L-selectin on neutrophils and P- and E-selectins on endothelial cells [1]. A firm attachment to the vascular surface is formed by shedding of L-selectin and increased expression of beta2-integrins on the neutrophil with ensuing binding of the beta2-integrins to intercellular endothelial adhesions molecules 1 and 2 (ICAM-1 and ICAM-2) on endothelial cells. Neonatal neutrophils, especially those found in preterm infants, appear to have decreased rolling and adhesion capabilities due to reduced L-selectin expression, L-selectin shedding, and beta2-integrin protein upregulation. An immature vascular endothelium with suboptimal P-selectin may also contribute to poor neutrophil rolling and adhesion [2, 3, 6].

Diapedesis and Basement Membrane Passage

Diapedesis, in which neutrophils migrate between endothelial cells and into the extravascular space, requires a cytoskeleton capable of deformability in addition to the beta2-integrin and ICAM interactions noted above. Furthermore, successful movement through the endothelial basement membrane is facilitated by neutrophil release of digestive enzymes, including elastase and collagenase [1, 2]. Neutrophils from newborns exhibit increased rigidity likely secondary to altered F-actin levels and abnormal membrane fluidity with resultant inability to modify cellular shape upon stimulation. Reduced elastase degranulation capabilities in preterm infants likely also affects passage through the endothelial basement membrane [2–4, 6].

Chemotaxis

The movement of neutrophils within the extravascular space across a gradient of chemotactic factors is termed chemotaxis. This cellular propulsion requires sensory receptors for various chemotactic peptide molecules. Impaired kinesis of newborn neutrophils results from not only reduced expression but decreased function of these receptors. Intracellular calcium plays an important role in chemotaxis following receptor stimulation and neutrophils within neonates appear to have suboptimal intracellular free calcium levels following activation [1, 2]. Term infants reach adult chemotactic abilities by approximately 2 weeks of age but preterm infants may not show significant improvement in chemotaxis until 2 months of age [6]. Sepsis, indomethacin administration, and intrapartum exposure to magnesium sulfate may further negatively affect chemotactic abilities [3, 7].

Phagocytosis

Upon entry into areas of inflammation neutrophils may encounter pathogens necessitating phagocytosis to aid in microbial clearance. Successful phagocytosis by neutrophils is aided by complement receptors, particularly the CR3 receptor, and Fc gamma receptors, which bind immunoglobulins as both complement and immunoglobulin are key contributors to opsonization. The ability to upregulate CR3 is decreased in both preterm and term infants. The expression of Fc gamma receptors in term infants appears to be comparable to adults but preterm neonates show significantly less expression of these receptors [6]. Interestingly, measurement of the phagocytic ability of neutrophils from term newborns was similar to adult controls but preterm neonates manifested greatly reduced phagocytosis when whole blood assays were used. The phagocytic defect observed in preterm infants can be improved by exposing the neutrophils to serum from adults or therapeutic immunoglobulin. Thus it appears that preterm and term neonates can show nearly normal phagocytic abilities if the focus of phagocytosis is opsonized appropriately [3].

Respiratory Burst

Following ingestion by phagocytosis, microorganisms are internalized into phagosomes, which then fuse with lysosomes and granules that contain proteolytic enzymes. These enzymes are next activated in the phagolysosome and digest the microorganism via generation of reactive oxygen species (using NADPH oxidase) and nitric oxide (using nitric oxide synthetase). Myeloperoxidase may additionally use the reactive oxygen species to create hypochlorous acid, which further assists in destruction in pathogens [1,3]. Under non-stressed conditions neutrophils from healthy newborns appear to show an intense respiratory burst response with increased reactive oxygen species generation. This elevated response is not sustained though at times of stress, including pulmonary or infectious insult, with neutrophils from ill neonates exhibiting suppressed respiratory burst. Healthy preterm newborns additionally show a reduced capacity to upregulate oxidative burst following bacterial stimulation. These neonatal respiratory burst deficiencies appear to persist throughout times of infection but will often normalize by 2 months of age in healthy infants [2–4].

Neutrophil Extracellular Traps (NETs)

Neutrophils also participate in a non-phagocytosis method of killing through the creation of neutrophil extracellular traps (NETs). NETs consist of DNA fragments and granule proteins extruded from neutrophils that form an extracellular scaffold of fibers with a high concentration of antimicrobial components with the ability to capture and destroy microbes. NET formation results from a cellular process that is separate from necrosis or apoptosis. The creation of NETs by both term and preterm infants is impaired and appears to further potentially increase their risk of infections [3, 4, 8].

CARD9 Deficiency

CARD9 deficiency is an autosomal recessive disorder due to mutations in caspase recruitment domain-containing protein 9 (CARD9), an intracellular myeloid signaling molecule downstream of C-type lectin and Toll-like receptors [19]. Mutations in CARD9 render the neutrophil defective against Candida and other serious fungal infections. Patients with CARD9 deficiency not unexpectedly present with a heightened susceptibility to fungal diseases including central nervous system and gastrointestinal tract infections with Candida, invasive Exophiala infections, and persistent mucocutaneous candidiasis. The invasiveness of the fungal disease is likely secondary to the killing defect of neutrophils and separates CARD9 deficiency from other disorders with chronic mucocutaneous candidiasis.

The deficit in fungal immunity is a result of the breakdown of the normal host defense. Normal host defense against Candida relies on opsonins, especially complement, and importantly the interaction of β-glucan and mannan residues on the surface of Candida with the C-type lectins expressed on the plasma membrane of myeloid cells to trigger an inflammatory response [20]. Activation of the C-type lectin or Toll-like receptors via recognition of fungal cell wall components results in phosphorylation of CARD9 and formation of a trimeric complex with B-cell lymphoma 10 (BCL10) and mucosa-associated lymphoid tissue lymphoma translocation gene 1 (MALT1). Formation of this trimeric CARD9-BCL10-MALT1 complex subsequently activates NF-ĸB and generates a pro-inflammatory response with IL-6 and IL-1β production that enhances antifungal, particularly anti-Candida, neutrophil function. CARD9 is also induces T-helper cells to produce interleukin-17 (IL-17), which plays a major role in the clearance of Candida [20]. Treatment of CARD9 deficiency relies on aggressive anti-fungal therapy in combination of with G-CSF and/or GM-CSF.

Chédiak–Higashi Syndrome

Chédiak–Higashi syndrome (CHS) is an autosomal recessive disorder that manifests with oculocutaneous albinism, neurologic dysfunction, abnormal coagulation, an elevated risk of hemophagocytic lymphohistiocytosis (HLH), and frequent infections with bacterial pathogens. A defect in the CHS1/LYST gene leads to abnormalities in lysosomal/vesicular transport and granulopoiesis. Giant azurophilic granules develop within patient neutrophils and other granulocytes and their appearance can assist in the diagnosis of patients [1]. Additionally, CHS neutrophils are deficient in granule proteases, suffer from delayed/incomplete degranulation and display faulty chemotaxis. [1, 21]. These functional neutrophil defects lead to recurrent and potentially serious cutaneous, respiratory tract, and mucous membrane bacterial infections. The lysosome and granule dysfunctions of the disorder lead to the clinical manifestations of albinism and coagulopathy as a result of improper transportation of melanosomes within melanocytes and reduction in platelet-dense bodies creating a platelet storage pool deficiency. Further hematological manifestations of the syndrome include neutropenia and a risk of development of hemophagocytic lymphohistiocytosis as the patient ages and enters the “accelerated phase” of the disorder. Patients are additionally at risk of developing progressive neurologic abnormalities, including ataxia, peripheral neuropathy, seizures, motor/sensory deficits, and cognitive decline [1, 22].

Diagnosis should be considered in those caring for neonates exhibiting albinism and frequent pyogenic infections. Examination of the infant’s blood smear may reveal absolute neutropenia and the presence of the classic large granules within all granulocytes, including neutrophils. Examination of fetal hair may reveal giant melanin granules. Definitive diagnosis is made via mutational testing for the CHS1/LYST gene. In those with a family history of the syndrome, prenatal diagnosis is additionally possible via analysis of chorionic villus or amniotic cells [1, 22].

Therapy is aimed at prophylactic administration of antibiotics, often trimethoprim-sulfamethoxazole, and aggressive treatment with antimicrobial therapy in those with proven or suspected infection. G-CSF administration may improve neutropenia. Patients often are referred to undergo hematopoietic stem cell transplantation (HSCT) prior to the development of hemophagocytic lymphohistiocytosis or devastating infections, but HSCT does not correct the neurologic deficits [1]. Treatment with ascorbic acid at high doses may improve the function of neutrophils in those with CHS and is reasonable to trial in patients [1, 23–25]

Chronic Granulomatous Disease

Chronic granulomatous disease (CGD) occurs with an estimated frequency of 1:200,000 live births and is due to mutations that disrupt the oxidative burst capacity of the nicotinamide adenine dinucleotide phosphate (NAPDH) oxidative complex in phagocytes [26]. Mutations in any of the genes (CYBB, CYBA, NFC1, NFC2, NFC4) that encode the five subunits (gp91phox, p22phox, p47phox, p67phox, p40phox, respectively) of NAPDH oxidase result in CGD. Defective oxidative burst results in the inability of neutrophils, monocytes and macrophages to kill many organisms, particularly catalase-positive bacteria such as Staphylococcus aureus, Burkholderia cepacia, Serratia marcescens, Nocardia, Samonella, and fungi such as Aspergillus.

Patients with CGD frequently present in infancy with the majority of patients presenting by childhood with severe or recurrent catalase-positive bacterial or fungal infections affecting the skin, lungs, liver, and lymph nodes. Fungal infections are the most common cause of death and carry a higher mortality risk than bacterial infections, although infections in CGD patients may be clinically underwhelming even with extensive disease [27]. Besides infections, patients with CGD are prone to inflammatory complications including granuloma formation and inflammatory bowel disease [28]. The dihydrorhodamine 123 (DHR) oxidation test can functionally assess the oxidative burst in clinically concerning patients. Gene sequencing can further confirm the disorder. Of note, patients with the X-linked form of CGD, due to mutations in CYBB, generally present with more severe disease as do patients with completely absent or extremely low residual superoxide production [29].

Treatment of CGD relies on aggressive treatment of active infections as well as prophylactic antimicrobials, e.g., trimethoprim-sulfamethoxazole, and antifungals, e.g., itraconazole [30]. Many patients also benefit from the use of interferon-gamma, although treatment with interferon-gamma is not universal [31]. Patients can be cured with hematopoietic stem cell transplant but the decision to proceed to transplant is based on the degree of disease severity and the quality of the donor match [32]. A limited number of patients have also been treated with gene therapy.

Glycogen Storage Disease Type 1b

Glycogen storage disease type 1b, also known as glucose-6-phosphatase deficiency or von Gierke disease, is an autosomal recessive condition most prominently associated with hypoglycemia and renal/hepatic abnormalities, but the disorder may also manifest neutropenia with dysfunctional neutrophil chemotaxis and respiratory burst capabilities [33, 34]. The majority of patients present with neutropenia prior to 1 year of age, although the neutropenia may be intermittent. The clinical impact of the neutropenia and neutrophil dysfunction is often clinically apparent in patients with the presence of perioral and perirectal infections as well as protracted diarrhea/colitis frequently reported [33]. A definitive diagnosis is made via genetic testing and G-CSF administration may reduce the frequency and severity of infections.