Neutrophil Extracellular Trap Formation in Newborn Infants at Risk for Inflammatory Syndromes
Conditions: Prematurity, Necrotizing Enterocolitis
Sponsor: University of Utah

Neutrophil Extracellular Trap Formation in Newborn Infants at Risk for 
Inflammatory Syndromes 
Protocol v 1, 1/15/2019 
PI:  Christian Yost, MD 
IRB# 119244 
Background 
Dysregulation of acute inflammation contributes to the pathogenesis of many neonatal and pediatric diseases 
including sepsis, acute respiratory distress syndrome (ARDS), neonatal chronic lung disease (nCLD), retinopathy of 
prematurity (ROP), and necrotizing enterocolitis (NEC).  
nCLD causes significant morbidity and mortality in the United States.  nCLD is a complication of preterm birth that 
results from prolonged mechanical ventilation required for chronic respiratory failure. It occurs in 30 - 50% of 
mechanically ventilated extremely low birthweight infants (ELBW) with respiratory distress. While surfactant 
therapy and pre- or postnatal steroids have decreased the severity of nCLD, this significant morbidity associated 
with preterm birth remains common in at – risk infants, with about 8,000 to 10,000 new cases occurring annually 
in the US. The mortality rate due to nCLD in ELBW infants remains high from 15 - 40%.  Furthermore, nCLD remains 
the most common cause of long-term hospitalization in neonates and is also associated with developmental 
delay.  Whether or not dysregulated NET formation contributes to the pathogenesis of nCLD remains unknown. 
ROP is the most common cause of pediatric blindness.  Dysregulated inflammation is also implicated in the 
pathogenesis of ROP leading to retinal detachment and blindness.  To date, no one has determined whether 
dysregulated NET formation contributes to ROP.   
NEC, predominantly a disease of premature infants, afflicts ≈ 5 - 10% of very low birth weight infants (< 1500 
grams), develops typically in the 3rd to 4th week of life and often results in mortality.  Infants born closer to term 
rarely develop NEC but those that do typically exhibit other risk factors such as ductal-dependent congenital heart 
disease or mesenteric ischemia.  For these patients, NEC occurs typically in the first week of life and can prove 
fatal.  Other sequelae of NEC include intestinal perforation, short gut syndrome, prolonged total parenteral 
nutrition with possible concomitant liver failure and a prolonged intensive care unit stay.  While the etiology 
remains unknown, risk factors of prematurity, enteral feeds, infection and intestinal ischemia predict an increased 
risk for NEC5. 
The final common pathogenic pathway for NEC appears to depend on a dysregulated, pro-inflammatory response 
to bacterial translocation across an immature or damaged gastrointestinal epithelial barrier.  Animal and human 
studies of NEC pathogenesis implicate inflammatory cytokines and phospholipids such as tumor necrosis factor 
alpha, Interleukin-8 and platelet activating factor (PAF).  One animal model also demonstrated that aortic 
lipopolysaccharide (LPS) infusion caused a NEC-like syndrome in rats 6.  Each of these biologically active 
inflammatory mediators activates the polymorphonuclear leukocyte (PMN)7, the primary effector cell of the acute 
inflammatory response to tissue injury or infection. 
Data from animal models suggest a prominent role of the PMN in the pathogenesis of NEC.  Musemeche et al. 
induced NEC-like disease in rats by intra-aortic injection of PAF8.  They then used vinblastine, a chemotherapeutic 
agent, to induce neutropenia in rats four days prior to intra-aortic injection of PAF.  The vinblastine-induced 
neutropenia protected against the clinical and pathologic manifestations of NEC-like disease.  

The role of the human PMN in the acute inflammatory response is well documented.  PMNs play a fundamental 
role in the innate immune response and are rapidly recruited to areas of injury or inflammation where they 
participate in bacterial phagocytosis and killing.  Disorders associated with a deficiency or impairment of PMN 
function (neutropenia, chronic granulomatous disease (CGD), leukocyte adhesion deficiency) predispose to 
infections with gram-negative and gram-positive bacteria9.    However, regulation of this potent component of the 
acute inflammatory response is imperative.  Disorders such as ARDS, ischemia/reperfusion injury, and rheumatoid 
arthritis appear to result from the dysregulation of the PMNs’ acute inflammatory response.  
Recently, neutrophils isolated from healthy adult donors were shown to undergo programmed cell death distinct 
from apoptosis and necrosis to form neutrophil extracellular traps (NETs).  NETs are extensive lattices of 
extracellular DNA and chromatin decorated with anti-microbial proteins and degradative enzymes such as 
myeloperoxidase and neutrophil elastase (NE).  NETs effect extracellular killing of bacteria and fungi.  Our 
laboratory recently characterized impaired NET formation as a novel innate immune deficiency of human newborn 
infants and showed that PMNs isolated from the cord blood of newborn infants, both term and preterm, 
demonstrated impaired NET formation and extracellular bacterial killing as compared to PMNs isolated from 
healthy adults.  However, the timing for developmental maturation of newborn infant PMN NET formation remains 
unknown.  Furthermore, while failure of NET formation contributes to the predisposition for and severity of 
bacterial and fungal infections associated with disorders such as CGD, dysregulated and inappropriately robust NET 
formation may lead to immune-mediated host tissue damage such as autoimmune small vessel vasculitis. 
Using data generated from the previous version of this IRB application (IRB_00039621 – Neutrophil Extracellular 
Trap Formation in Newborn Infants at Risk for Necrotizing Enterocolitis – Closed on 11/8/2018), we recently 
discovered a new regulatory mechanism governing NET formation in the first 2 weeks following birth for neonates, 
whether term or preterm.  Using cord blood samples as well as samples of peripheral blood taken serially from 
neonates over the first 2 months of life, we recently identified a family of NET-Inhibitory Peptides (NIPs) which 
circulate in the fetus and neonate and potently inhibit PMNs from forming NETs.  These NIPs inhibit NET formation 
despite stimulation with many NET-generating agonists like LPS, PMA, live gram+ or gram- bacteria, heme, or 
viruses.  We further examined the use of these NIPs in the setting of in vivo sepsis.  We found that pretreatment 
with the NIPs increased survival in the cecal ligation and puncture model of polymicrobial sepsis by 40-60%, with 
the survival advantage most likely due to decreased NET-mediated inflammatory tissue damage.  With these 
published results (Yost CC, et al., JCI 2016), we are now funded by the NIH to evaluate the use of NIPs as 
therapeutic agents in preclinical models of neonatal sepsis.  These studies will determine regulatory pathways 
governing NIP expression in the fetus and newborn, NIP mechanism of action, and efficacy in neonatal sepsis.        
We hypothesize that NET formation contributes to the pathogenesis of inflammatory syndromes associated with 
sequelae of prematurity including sepsis, nCLD, ROP, and NEC by inappropriately releasing degradative proteins 
and tissue destructive enzymes into the inflammatory milieu of the premature infant.  We further hypothesize that 
NIPs may provide protection against NET-associated inflammatory tissue damage. 
Study Purposes and Objectives: 
The following study is best described as a prospective, in vitro longitudinal cellular biology study of LPS/PAF-
stimulated PMNs isolated from the cord and peripheral blood of premature infants at risk for inflammatory 
sequelae associated with prematurity (including neonatal Chronic Lung Disease (nCLD), early and late onset sepsis, 
necrotizing enterocolitis (NEC), and retinopathy of prematurity), and from term infants not considered at risk for 
such sequelae. In the specific case of NEC, we will also assay for NET formation in gastrointestinal tissue samples 
obtained at the time of surgery for severe NEC in enrolled prematurely born infants if that occurs. Such tissue 
samples will not be collected for other inflammatory syndromes of prematurity.  
These studies are the first of their kind and aim to answer important questions such as the impact of dysregulated 
NET formation in the pathogenesis of these inflammatory sequelae of prematurity, the regulatory mechanisms 

governing NET formation in newborn infants, the role of our newly discovered NET-Inhibitory Peptides (NIPs) as 
endogenous regulators of NET formation, and their potential use as novel therapeutic agents for dysregulated NET 
formation. 
Inclusion Criteria: 
Eligible Patients 
  
1. Preterm infant patients delivered at UUMC and hospitalized in the NICU who are ≤ 1500 grams or < 30 weeks 
gestational age at birth 
  
Cord blood sample obtained at delivery 
  
Peripheral blood sample obtained during IV start under anesthesia at any elective surgeries during the first year of 
life 
  
 
  
2. Term infants delivered at UUMC without complication, either via cesarean section or vaginal delivery. 
  
Cord blood sample obtained at delivery 
  
Heel-prick blood sample obtained at 24 hours of life 
Exclusion criteria: 
None 
Study Procedures: 
Recruitment: We will screen charts through a waiver of authorization for potential participants within the 
University of Utah covered entity.  Eligible participants identified through the waiver of authorization will be 
identified once they are admitted to the hospital for clinical management, with the expectation of delivery during 
the current hospital admission.    
Inclusion/exclusion criteria will be reviewed with the patient’s chart and then approached once initial eligibility 
criteria have been verified.  
Pregnant women admitted to labor and delivery and who meet the eligibility criteria for term deliveries without 
complication will be identified and approached about the study prior to delivery. 
Pregnant women admitted to labor and delivery in an emergent manner and who meet the eligibility criteria for 
preterm delivery at <30 weeks gestational age may be approached prior to delivery, when deemed appropriate by 
the patient’s care team. 
Consent: For consent of healthy term neonates, all mothers will be approached after admission to the Labor and 
Delivery Unit at UUMC.  Informed consent for their neonate's participation in our study will be sought by Dr. 
Yost.  If the delivery is precipitous and cord blood is collected, the families of potential healthy term neonate 
participants may also be approached after the fact but within 24 hours after delivery if appropriate.  It is 
anticipated that this will be the exception and not the rule.  Furthermore, any umbilical cord blood collected will 
be used and tested for any clinical indications before being considered for use in research for this project.  Also, 

research related activities connected with this study will not begin until after consent has been obtained from the 
family of the newborn. 
For consent of preterm neonates born at less than 30 weeks gestation, all mothers admitted to the Labor and 
Delivery Unit at UUMC and meeting inclusion criteria will be approached for consent prior to delivery by Dr. Yost or 
his research team.  In each case, the obstetrical clinical care team will determine whether initiating the consent 
process prenatally is appropriate.  The family's physical and emotional state prior to delivery will be considered in 
this determination.  If appropriate, Dr. Yost or his research team will consent for study participation prenatally and 
proceed with use of the umbilical cord blood at that moment.  If not deemed appropriate, Dr. Yost's research team 
will collect the umbilical cord blood and approach the family for informed consent within 24 hours after 
delivery.  Furthermore, any umbilical cord blood collected will be used and tested for any clinical indications before 
being considered for use in research for this project.  Also, research related activities connected with this study will 
not begin until after consent has been obtained from the family of the newborn.   
Phlebotomy:  
Patient Group #1:  Prematurely born infants in the UUMC and PCH NICUs 
After delivery, cord blood will be drawn from the ligated umbilical cord and placenta.  For premature deliveries of 
infants weighing less than 1500 grams, it is estimated that only 5 – 10 mL of cord blood will be obtained.  The 
resuscitation and treatment of a premature infant often requires cord blood specimens for such clinical tests as 
arterial and venous cord blood gases and blood type and crossmatch in preparation for probable future blood 
transfusions.  These specific clinical cord blood samples will be collected before study blood collection.   The 
residual cord blood will be dripped into sterile 8 mL EDTA-anticoagulated vacutainers, labeled with a study 
generated patient identifier and transported by Dr. Yost or his laboratory technicians to his lab in the Eccles 
Institute of Genetics for immediate PMN isolation and sample stabilization.  This rapid isolation and sample 
stabilization is necessary given the short life of PMNs in vitro and the potential for PMN activation after isolation.  
The family will then be approached regarding informed consent as discussed below.  If informed consent is 
obtained, preparations will be made to obtain 3 serial 0.5 ml samples of peripheral blood during the infant’s NICU 
stay.  Samples will be collected on the routine lab draw day that most closely correlates with the following days of 
life (DOL) #:  3, 7, and 14 if still in the NICU at either UUMC or PCH. 
Longitudinal samples will be collected into a 0.5 ml EDTA-anticoagulated microtainers and processed in an identical 
manner to the cord blood already collected.  These serial samples will only be collected during routine clinical 
laboratory draws occurring around the scheduled DOL.  No new lines, IVs or heel stick wounds will be started or 
made for the sole purpose of drawing the scheduled study sample. 
For the small subset of at-risk infants who develop NEC requiring surgery at PCH, whether born at UUMC or 
transported to PCH from a referring institution, we will also obtain fixed and paraffin-embedded gastrointestinal 
tissue samples at the time of surgery for analysis of NET formation.  These samples will be obtained by the 
pediatric surgical team at the time of exploratory laparotomy and processed by the diagnostic pathology service at 
PCH. For those infants transported to PCH with surgical NEC from referring institutions, informed consent will be 
requested upon admission to PCH NICU.  If informed consent is obtained, the samples would be processed and 
transferred by Dr. Yost to his laboratory for further analysis.  
Patient Group #2:  Healthy term infants born at UUMC 
After delivery, cord blood will be drawn from the ligated umbilical cord and placenta.  For term deliveries at 
UUMC, it is estimated that approximately 15 - 25 mL of cord blood can be routinely obtained without 
compromising needs for possible clinical studies such as infant blood-typing.  Samples will be dripped into sterile 8 

mL EDTA-anticoagulated vacutainers, labeled with a study generated patient identifier and transported by Dr. Yost 
or his laboratory technicians to his lab in the Eccles Institute of Human Genetics for immediate PMN isolation and 
sample stabilization.  Again, this rapid isolation and sample stabilization is necessary given the short life of PMNs in 
vitro and the potential for PMN activation after isolation. 
The family will then be approached regarding informed consent as discussed below.  If informed consent is 
obtained, preparations will be made to obtain one additional 1 ml sample of peripheral blood during the infant’s 
heel stick wounding for the 24-hour metabolic screening test performed on all infants before DOL #3 and before 
hospital discharge.  The sample will be collected into 2 0.5 ml EDTA-anticoagulated microtainers and processed in 
an identical manner to the cord blood already collected.  This additional sample will only be collected during the 
routine 24 – 48-hour metabolic screening laboratory draw.  No new lines, IVs or heel stick wounds will be started 
or made for the sole purpose of drawing the scheduled study sample. 
Experimental Procedures:          
After parental consent is obtained, PMNs will be isolated via positive immunoselection against CD15 antigen 
expression as previously described.  PMNs will be stimulated with PAF or LPS and analyzed for NET formation via 
live cell imaging, quantitative extracellular DNA content and quantitative neutrophil elastase activity.  For NEC 
tissue samples, NET formation will be qualitatively assessed via immunohistochemistry.  NET-Inhibitory Peptide 
concentrations in blood plasma will be assessed via quantitative Western blotting and/or HPLC/MS2 assays. 
Live Cell Imaging:  
Isolated PMNs will be allowed to adhere to lysinated glass coverslips and stimulated with PAF [10 nM] or LPS [100 
ng/ml] for 1 – 4-hour time points.  Cell impermeable and cell permeable intercalating DNA dyes will be used to 
differentially stain NET-associated, extracellular DNA and intracellular nuclear DNA.  Qualitative assessment of NET 
formation will be captured and recorded via confocal microscopy. 
Quantitative extracellular DNA Content Assay:  
Supernatants following PAF/LPS stimulation and confocal imaging will be treated with DNase (66 U/ml for 15 
minutes) to break up NETs and gently removed and analyzed for total DNA content.  Supernatant DNA 
concentrations will be quantified by incubating the samples with Sytox Orange (1mM) and comparing the values 
against a concentration curve constructed with varying concentrations of a known DNA standard using fluorimetry 
with filter settings of 530(excitation)/ 645(emission) (BioTek Synergy HT). 
Quantitative Myeloperoxidase (MPO)-DNA ELISA:  
NET-associated MPO activity will be determined at selected time points as described.  Briefly, the incubation 
medium will be gently removed and replaced with medium containing DNase (66 U/ml) for 15 min.  A capture 
antibody against MPO will be employed as part of a MPO-DNA ELISA to provide quantitative levels of NET 
formation.  A detection antibody against human DNA will serve as the detection antibody. 
Quantitative NIP Concentration in Human Plasma: 
Plasma samples will be obtained from whole blood samples via centrifugation.  The aliquots of the plasma will then 
be run on Tris-Tricine precast gels to resolve proteins and peptides with a molecular weight between 1 and 30 
kD.  A standard curve with increasing concentration of synthesized NIPs will be run on the same gel and 
concentration will be determined by comparing the fluorescent intensity of the plasma band to those of the 

standard curve.  Alternatively, we are developing a HPLC/MS2 assay which will use a synthesized "heavy" variant of 
NIPs to quantify NIP concentration in plasma compared to a known amount of spiked "heavy" NIP.    
Qualitative assessment of NET formation in NEC tissue specimens via immunohistochemistry: 
NEC tissue specimens processed by pediatric pathology at PCMC and embedded in paraffin will be transported to 
Dr. Yost’s laboratory and analyzed for NET formation via immunofluorescence.  Surgical samples will be sectioned 
at 5 – 10 µm thickness.  NETs will be visualized by examining the colocalization of antibodies against DNA, histones, 
and neutrophil elastase. 
Data Analysis: 
NET formation as assessed qualitatively via live cell imaging and immunohistochemistry and quantitatively via 
extracellular DNA quantitation and neutrophil elastase activity assays.  
We anticipate enrolling 40 prematurely born infants in patient group # 1, 20 healthy term newborn infants in 
patient group # 2 during this study.  
Statistical comparisons for the quantitative assays of extracellular DNA and NE activity will be made via one-way 
ANOVA with post hoc Pairwise Multiple Comparisons made via the Holm-Sidak method.