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Original Article
Oral antibiotics increase blood neutrophil
maturation and reduce bacteremia and
necrotizing enterocolitis in the immediate
postnatal period of preterm pigs
Duc Ninh Nguyen
1,
*, Eva Fuglsang
2,*
, Pingping Jiang
1
,
Malene M Birck
1
, Xiaoyu Pan
1
, Shamrulazhar BS Kamal
3
,
Susanne E Pors
2
, Pernille L Gammelgaard
1
, Dennis S Nielsen
3
,
Thomas Thymann
1
, Ofer Levy
4
, Hanne Frøkiær
2
and Per T Sangild
1,5
Abstract
Immature immunity may predispose preterm neonates to infections and necrotizing enterocolitis (NEC). Intravenous
antibiotics are frequently given to prevent and treat sepsis, while oral antibiotics are seldom used. We hypothesized that
oral antibiotics promote maturation of systemic immunity and delay gut bacterial colonization and thereby protect
preterm neonates against both NEC and bacteremia in the immediate postnatal period. Preterm pigs were given formula
and administered saline (CON) or broad-spectrum antibiotics orally (ORA) or systemically (SYS) for 5 d after birth.
Temporal changes in blood parameters and bacterial composition in the intestine, blood and immune organs were
analyzed. Newborn preterm pigs had few blood neutrophils and a high frequency of progenitor cells. Neutrophils
gradually matured after preterm birth with increasing CD14 and decreasing CD172a expressions. Preterm neutrophil
and monocyte TLR2 expression and TLR2-mediated blood cytokine responses were low relative to adults. ORA pigs
showed enhanced blood neutrophil maturation with reduced cell size and CD172a expression. Only ORA pigs, but not
SYS pigs, were protected from a high density of gut Gram-positive bacteria, high gut permeability, Gram-positive bac-
teremia and NEC. Neonatal oral antibiotics may benefit mucosal and systemic immunity via delayed gut colonization and
enhanced blood neutrophil maturation just after preterm birth.
Keywords
Antibiotics, neonatal immunity, neutrophils, preterm pig, TLR2
Date received: 20 August 2015; revised: 29 September 2015; accepted: 9 October 2015
Introduction
Neonatal immunity depends, to a large degree, on
innate cells and soluble mediators for protection
against environmental antigens and pathogens.
1,2
At
birth, the innate immune system is immature,
3
and
blood neutrophils and monocytes develop during late
gestation and after birth, reflected by their increased
expression of the surface receptors CD14, MD2,
TLR2, and TLR4.
1,4–6
In pigs, these markers have simi-
lar trends during cell maturation,
1,4,5
whereas CD172a
expression decreases postnatally.
7
Neutrophils also
1
Section of Comparative Pediatrics and Nutrition, Department of
Veterinary Clinical and Animal Sciences, University of Copenhagen,
Frederiksberg, Denmark
2
Department of Veterinary Disease Biology, University of Copenhagen,
Frederiksberg, Denmark
3
Department of Food Science, University of Copenhagen, Frederiksberg,
Denmark
4
Division of Infectious Diseases, Department of Medicine, Boston
Children’s Hospital and Harvard Medical School, Harvard, MA, USA
5
Department of Pediatrics and Adolescent Medicine, Rigshospitalet,
Copenhagen, Denmark
Corresponding author:
Per T Sangild, Section of Comparative Pediatrics and Nutrition,
Department of Veterinary Clinical and Animal Sciences, University of
Copenhagen, Dyrlægevej 68, DK-1870 Frederiksberg C, Denmark.
Email: pts@sund.ku.dk
*DNN and EF contributed equally to this work.
Innate Immunity
2016, Vol. 22(1) 51–62
!The Author(s) 2015
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DOI: 10.1177/1753425915615195
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decrease in size during maturation,
8
and
monocytic CD14 expression increases when activated
by bacteria.
9
In preterm infants, insufficient time for immune mat-
uration likely plays a key role in their susceptibility to
infection, sepsis and necrotizing enterocolitis (NEC).
10–12
Compared with term, preterm infants have markedly
fewer leukocytes, and lower expression of CD14, MD2,
TLR2 and TLR4 in blood neutrophils and monocytes.
1,13
NEC and sepsis have been shown to be associated with
neutropenia, monocytopenia, thrombocytopenia and/or
suppression of erythropoiesis,
14–17
although it remains
unclear whether systemic immunity is functionally con-
nected with these conditions.
NEC pathogenesis is incompletely understood,
although premature birth, excessive bacterial coloniza-
tion and aggressive enteral feeding are important pre-
disposing factors.
12
Breast milk is believed to protect
against NEC, relative to infant formula,
18,19
but is
often absent during the first week after preterm birth.
Modulation of initial gut colonization by anti-, pre-
and probiotics may reduce NEC risk but the optimal
efficacy, time and dose of such treatments are
unclear.
20–25
Although antibiotics have been reported
to decrease NEC incidence in several small studies,
25
there is a well-justified reluctance to use prophylactic
antibiotics owing to insufficient mechanistic evidence
for benefits and risk of antibiotic resistance. In add-
ition, it is important that prolonged postnatal adminis-
tration of systemic antibiotics is associated with altered
gut bacterial colonization and increased risk of NEC in
preterm infants.
26
The preterm pig is the only described animal model
that incorporates the normal clinical signs of prematurity
(e.g. respiratory defects, hypothermia, dysmetabolism,
elevated infection risk) and spontaneously develops
NEC following feeding with infant formula during the
postnatal period.
27
Preterm pigs also allow parenteral
and enteral feeding protocols, repeated blood sampling
and detailed sample collection from the gut lumen and
mucosa to monitor parameters related to infection and
NEC.
28
We have previously shown that broad-spectrum
antibiotics (ampicillin, gentamicin and metronidazole)
provided simultaneously via the oral and systemic
routes for 5 d completely prevented NEC in formula-
fed preterm pigs.
24
In this study, it was not clear if sys-
temic immunity was affected or if both oral and systemic
administration was required for NEC protection.
Without antibiotics and standard clinical care, gut colon-
ization rapidly progresses to reach a colonic bacterial
density of 10
10
–10
11
bacteria after birth.
24
Preterm new-
borns are susceptible to translocation of bacteria, or bac-
terial products, from the gut and other epithelial surfaces
into the blood stream.
1,2
Conversely, oral antibiotics may
delay neonatal colonization of the gut and thereby main-
tain the immature intestine in a state that more resembles
the bacteria-deficient in utero condition. In turn, this may
facilitate immune maturation during the critical period
just after preterm birth.
We hypothesized that systemic immunity is imma-
ture in newborn preterm pigs with functional deficits
in specific leukocyte populations and that leukocyte
maturation occurs during the first few days after pre-
term birth when enteral feeding volumes are still low
and NEC symptoms are absent. We speculate that only
oral, not systemic, administration of antibiotics
enhances neonatal blood leukocyte maturation, delays
gut colonization and thereby protects against bacter-
emia and formula-induced NEC.
Materials and methods
Experimental animal procedures
The animal protocol was approved by the Danish
National Committee of Animal Experimentation.
Cesarean-delivered preterm pigs (105–106 d of gesta-
tion, 90–92%) from three sows received parenteral
nutrition (4–6 ml/kg/h) via umbilical catheters and min-
imal enteral nutrition (3 ml/kg/6 h) with formula via
orogastric tubes for the first 2 d, followed by 2 d with
total enteral nutrition (15 ml/kg/3 h) with formula. The
composition of the formula was specifically designed
for the piglets’ need. Pigs were randomized into three
groups: those receiving saline (CON, n¼17); those
receiving broad-spectrum antibiotics via the oral route
(ORA, n¼18); or those receiving antibiotics via the
systemic route via umbilical catheters (SYS, n¼17).
Antibiotics (ampicillin: 30 mg/kg three times daily; gen-
tamicin: 2.5 mg/kg twice daily; metronidazole: 10 mg/kg
three times daily) were given in identical doses to ORA
and SYS pigs based on the commonly used i.v. anti-
biotic regimes used for pediatric patients with suspected
NEC or sepsis. On d 5, pigs were euthanized and five
gastrointestinal regions (stomach, proximal, middle
and distal small intestine, and colon) were macroscop-
ically evaluated for pathological changes by a NEC
scoring system (score 1–6), indicative of the severity
of inflammation and necrosis. An animal was desig-
nated as NEC-positive when a minimum score of 3 in
at least one region was observed. Further details of pig
handling, housing, feeding, NEC scoring and clinical
assessment were described previously.
24,29
Other spe-
cific details of the protocol are described in the
Supplementary Material.
Blood was collected on d 1 (cord), 2 and 3 (umbilical
catheter), and at euthanasia (d 5, cardiac puncture).
For bacterial characterization, samples of blood,
spleen and femur-derived bone marrow were aseptically
collected at euthanasia. Furthermore, cord blood from
four Cesarean-delivered term pigs (116–117 d of gesta-
tional age) and blood from 11 healthy adult pigs (1yr
old) were collected for comparison with blood from
preterm pigs.
52 Innate Immunity 22(1)
Blood phenotyping
Blood cell counting was analyzed by an automatic cell
counter (Advia 2120i Hematology System; Siemens,
Munich, Germany). For further phenotyping, whole
blood (100 ml) was used and erythrocytes were lysed
by 1 BD FACS Lysing solution (BD Biosciences,
Franklin Lakes, NJ, USA). Lysed blood was washed
by PBS supplemented with FBS and NaN
3
. Fc recep-
tors on leukocytes were then blocked by porcine serum
[10 min, room temperature (20C), in the dark] prior to
leukocyte staining with the following Abs: CD14–FITC
(MIL2; AbD Serotec, Kidlington, UK), CD172a-PE
(74-22-15A; BD Biosciences), TLR2–AF647 (CD282,
11G7; AbD Serotec). IgG2b-FITC (AbD Serotec) and
IgG2b-PE (eBioscience, San Diego, CA, USA) were
used as isotype controls. Cells were incubated with sur-
face Abs (15 min, room temperature, in the dark), and
washed twice prior to fixation in 4% formalin. Cells
were analyzed on a BD FACS Canto II flow cytometer
(BD Biosciences). Staining profiles were analyzed using
FlowJo (Tree Star, Inc., Ashland, OR, USA).
Gating strategy was performed as previously
described in pig leukocytes,
30
and further described in
the Supplementary Material and Supplementary
Figures S1 and S2. Briefly, based on CD172a, CD14
and side scatter (SSC), neutrophils were
CD172a
+
CD14
+
SSC
high
, and monocytes were
CD172a
+
CD14
+
SSC
medium
. Small cells (low forward
scatter; FSC) with variable granularity were judged to
be progenitor cells. TLR2 expression was low in both
monocytes and neutrophils in blood from preterm pigs.
Blood stimulation with TLR2 and TLR4 agonists
At Cesarean section in each of three separate litters, a
pooled cord blood sample from all preterm pigs and
sow blood were collected. Blood was stimulated with
TLR agonists (Invivogen, San Diego, CA, USA):
freeze-dried heat-killed Listeria monocytogenes
(HKLM, TLR2 agonist, 10
2
–10
6
cells/ml), Pam
3
CSK
4
(bacterial lipopeptide, TLR1/2 agonist, 10
4
–1 ng/ml)
and LPS (TLR4 agonist, 10
2
–10
2
ng/ml). After stimu-
lation at 37
o
C, 5% CO
2
for 5 h, blood was diluted five-
fold with RPMI 1640 medium (Life Technologies,
Carlsbad, CA, USA), centrifuged at 2500 gat 4
o
C for
10 min. Supernatants were analyzed for IL-6 and TNF-
aby ELISA (R&D Systems, Abingdon, UK).
TLR2 gene expression in whole blood
RNA from blood samples was extracted using
MagMAX 96 total RNA Isolation Kit (Life
Technologies) and converted into cDNA as previously
described.
31
Quantitative real-time PCR (qPCR) was
performed using TaqMan Universal Master Mix II
(Life Technologies) to quantify TLR2 gene expression
in blood on d 1 and 3. Primers of TLR2 and the
housekeeping gene (b2 microglobulin; B2M)
were designed by PrimerExpress sofware (Applied
Biosystems, Foster City, CA, USA) and synthesized
by Applied Biosystems: TLR2 (F:
TCGAAAAGAGCCAGAAAACCAT and R:
CTTGCACCACTCGCTCTTCAC), and B2M (F:
ATCCGCCCCAGATTGAAATT and R:
TGCTCCGCGTTCATCTTCT). Relative gene expres-
sion of TLR2 was calculated by normalization to levels
of the reference gene B2M.
Bacterial adherence to intestinal mucosa
by fluorescence in situ hybridization
Bacterial abundance on three intestinal regions (prox-
imal, middle and distal) was evaluated by fluorescence
in situ hybridization (FISH) on a paraffin-embedded
cross section (3 mm) using Alexa fluor 555-label oligo-
nucleotide probes (Eurofins MWG Operon, Ebersberg,
Germany) targeting the 16S rRNA of general bacteria
(50-GCTGCCTCCCGTAGGAGT-30),
32
and visualized
by an fluorescence microscope. The tissue sections were
scored from 0 to 6 based on the fluorescence signal,
where 0 ¼no bacteria, 1 ¼very few bacteria, 2 ¼few
spread bacteria, 3 ¼bacteria spread in the whole
tissue section, 4 ¼minor colonies spread in the whole
tissue section, 5 ¼numerous larger colonies, and
6¼widespread overgrowth with large colonies. Values
are means SEM across intestinal regions.
Bacterial identification and quantification
Blood, homogenized spleen and bone marrow were cul-
tured on blood agar (5% bovine blood supplemented-
Blood Agar Base CM0055; Oxoid, Basingstoke, UK) at
37
o
C for 24 h. After enumeration, different bacterial
colonies were counted, isolated, transferred and incu-
bated in Brain–Heart Infusion Broth (Difco, Brøndby,
Denmark) at 37
o
C for 24 h. Bacterial identification
from cultured pure isolates were performed by a
MALDI-TOF Mass spectrometer (Vitek MS,
Biome
´rieux, France).
33
Total DNA from colon contents was extracted and
total bacterial load was quantified by qPCR (see
Supplementary Material). Colonic prokaryotic micro-
biota composition was determined by tag-encoded 16S
rRNA gene MiSeq-based (Illumina, San Diego, CA,
USA) high-throughput amplicon sequencing.
34,35
Intestinal permeability
Three h prior to euthanasia, pigs received an oral bolus of
5% lactulose and 5% mannitol (15 ml/kg). The ratio of
urinary lactulose and mannitol levels at euthanasia was
determined as the indicator of intestinal permeability,
24
as
mannitol and lactulose are markers of transcellular
absorption and paracellular permeability, respectively.
Nguyen et al. 53
Statistics
Cell count comparisons among newborn preterm, term
and adult pigs, NEC and FISH scores, and bacterial
load were analyzed by the Kruskal–Wallis test (JMP
10; SAS Institute, Cary, NC, USA). NEC incidence
was analyzed by Fisher’s exact test, and cytokines
from blood assays were tested in a linear mixed
model using pig (preterm or adult) and agonist concen-
tration as fixed factors (JMP). Other parameters were
fitted to a linear mixed model with treatment, day and
litter as fixed factors, and gender and pig code as
random factors using the ‘lmer’ function (R; www.R-
project.org).
36
The significance of treatment and day
was tested by the Tukey test using an ANOVA func-
tion. Differences among groups on each day were tested
using the ‘glht’ function in the package ‘multcomp’.
37
Results
Oral antibiotics prevent NEC
No pigs from the ORA group developed NEC lesions
(incidence 0%, 0/16), compared with 63% (10/16)
in CON and 59% (10/17) in SYS pigs (P<0.001;
Figure 1A). No difference in NEC incidence was
observed between CON and SYS pigs. The average
NEC severity score of five gastrointestinal regions was
lower in ORA than SYS and CON pigs (P<0.01;
Figure 1B). NEC lesions were relatively mild in both
CON and SYS pigs, as the lesions were present mainly
in the colon with limited differences in the small intes-
tine (Figure 1C, D).
Immature blood leukocytes in newborn preterm pigs
Newborn term and adult pigs had higher concentra-
tions of total blood leukocytes (2.2- and 4.3-fold),
neutrophils (7.1- and 9.7-fold), erythrocytes (1.5- and
1.2-fold) and hemoglobin (1.4- and 1.2-fold) than pre-
term pigs at birth (all P<0.001; Table 1). Preterm
blood also had lower counts of lymphocytes, monocytes,
eosinophils and basophils relative to adult blood
(P<0.001). Flow cytometric analysis showed that progeni-
tor cells comprised 20–30% of total leukocytes in preterm
pigs, in contrast with the negligible amounts observed in
term and adult pigs (Supplementary Figure S3). These
findings suggest an immature state of innate immune
cells in preterm pigs, relative to term and adult pigs.
Preterm blood neutrophil and monocyte
development during the first 3 d of life
Leukocyte development was characterized from d 1 to
d3 in preterm pigs (all treatment groups were combined
owing to similar developmental trends), prior to the
increases in enteral feeding volume and before any clin-
ical signs of NEC.
38
Total leukocyte and neutrophil
counts peaked on d 2 and returned to basal levels on
d 3, whereas other cell counts remained constant during
d 1–3 (Supplementary Figure S4).
The expression of CD172a, CD14 and TLR2 and
cell size (reflected by FSC) were used as markers of
maturation and development of neutrophils and mono-
cytes (Figure 2A–C). CD14 intensity and frequency of
CD14
high
cells increased, and frequency of CD14
low
cells decreased (P<0.001) in both neutrophils and
monocytes on d 2–3, relative to d 1. In parallel, neu-
trophil and monocyte CD172a expression decreased
over time (P<0.001), similar to their postnatal devel-
opment in term pigs.
7
Over time, neutrophil size
decreased, whereas monocyte granularity increased
(P<0.001). These changes indicate a gradual matur-
ation and development of blood neutrophils and
NEC vs No NEC
Average NEC score
NEC score-colon
NEC score-small intestine
100
80
60
40
20
0
0
0
0.0
CON SYS ORA
0.5
1.0
1.5
2.5
2.0
1
2
3
4
5
1
2
3
NEC serverity, score
NEC serverity, score
NEC serverity, score
a
a
a
a
a
b
b
ab
37%
63%
41%
59%
NEC
No NEC
100%
Incidence of
NEC vs no NEC (%)
(a)
(b)
(c)
(d)
Figure 1. Pathological evaluation. (a) NEC incidence, (b) aver-
age NEC severity score throughout the gastrointestinal tract, and
average NEC score in the (c) colon and (d) small intestine
(n¼12–17 per group). Values (means SEM) in the same region
not sharing common letters are significantly different (P<0.05).
54 Innate Immunity 22(1)
monocytes in preterm pigs. In contrast, TLR2 intensity
was low and did not change in both cell types during d
1–3, suggesting impaired development of TLR2
responses. TLR2 presence was confirmed by qPCR
analysis of blood RNA. Of note, the frequency of pro-
genitor cells decreased over time (Figure 2D).
Impaired TLR2-mediated cytokine production
from blood leukocytes in preterm pigs
To assess the functional correlates of TLR2 expression,
we measured TLR2 agonist-induced pro-inflammatory
cytokine production in whole blood. TLR2-mediated
TNF-aand IL-6 production (via Pam
3
CSK
4
and
HKLM cells) were markedly lower in newborn pre-
term vs. adult pig blood (P<0.01; Figure 3A–D).
Preterm blood leukocytes did not produce IL-6 at
any of the TLR2 agonist concentrations, in contrast
to the 200–500 pg/ml IL-6 found in adult blood.
TLR2 agonist-induced TNF-alevels in preterm
blood were either negligible or less than half of that
in adult blood. These findings suggest a negligible
expression and poor function of surface TLR2 in
innate blood leukocytes of preterm pigs. In contrast,
TLR4-mediated TNF-aand IL-6 production in new-
born preterm blood was more similar to that in adult
blood (Figure 3E, F).
Oral antibiotics enhance neutrophil maturation
ORA pigs showed more advanced neutrophil matur-
ation, shown by lower cell size and CD172a expression
on d 2 and 3, compared with CON and SYS pigs
(P<0.05; Figure 4A). In parallel, progenitor cells
showed a strong trend for a lower percentage in ORA
pigs on d 3 (P¼0.05; Figure 4B). These findings sug-
gest that oral administration of antibiotics accelerates
blood neutrophil maturation. In contrast, monocytes in
CON pigs were more granular (P<0.05) and tended to
have higher CD14 expression than ORA and SYS pigs
on d 3 (P¼0.08; Figure 4B), indicating more inflam-
matory monocytes in CON pigs.
Effects of antibiotics and NEC on blood
leukocytes at euthanasia on d 5
At euthanasia, ORA pigs had lower monocyte number
and granularity relative to CON pigs (P<0.05;
Supplementary Figure S5). Additionally, healthy pigs
without NEC on d 5 also had greater total leukocyte
and lymphocyte counts, and lower monocyte granular-
ity than pigs with NEC (P<0.05; Supplementary
Figure S5).
Oral antibiotics reduce bacterial adherence, gut
colonization, gut permeability and bacteremia
The FISH scores in the small intestine, reflecting the
relative abundance of bacteria that adhere to the intes-
tinal mucosa, were highest (2.4 0.3) in CON, followed
by SYS (1.9 0.3) and lowest in ORA pigs (1.1 0.3;
P<0.01). Intestinal permeability shown by the urinary
lactulose/mannitol ratio did not differ in pairwise group
comparisons but was significantly reduced in ORA
pigs, relative to the combined value from CON and
SYS pigs (0.027 0.022 vs. 0.094 0.016; P<0.05).
ORA pigs had 2 log (100-times) lower bacterial load
in colon contents than CON and SYS pigs (P<0.05
and 0.01; Figure 5A). In blood, bacteria were not
detected in ORA pigs and were most abundant in
CON pigs. In bone marrow, ORA pigs showed mark-
edly reduced bacterial load relative to the other two
groups (Figure 5B; P<0.01). There was no significant
difference in bacterial load among the three groups in
the spleen although numerically, values remained
lowest in the ORA group.
Gram-positive bacteria (Enterococcus,Clostridium
and Staphylococcus spp.) were predominant in
the colon content of CON pigs, whereas most ORA
pigs were dominated by Gram-negative bacteria
Table 1. Hematological profile of newborn preterm (n¼51), newborn term pigs (n¼4) and healthy adult pigs (n¼11).
Hematological parameters Preterm newborn pigs Term newborn pigs Adult pigs P-value
Total leukocytes (10
9
/l) 2.61 0.35
a
5.70 1.47
b
11.48 0.72
c
<0.001
Neutrophils (10
9
/l) 0.50 0.03
a
3.55 1.08
b
5.02 0.37
b
<0.0001
Lymphocytes (10
9
/l) 1.98 0.08
a
2.06 0.41
a
5.33 0.54
b
<0.0001
Monocytes (10
9
/l) 0.07 0.01
a
0.04 0.01
a
0.44 0.05
b
<0.0001
Basophils (10
9
/l) 0.018 0.004
a
0.01 0.00
a,b
0.070 0.013
b
<0.0001
Eosinophils (10
9
/l) 0.030 0.003
a
0.03 0.02
a,b
0.37 0.09
b
0.0002
Erythrocytes (10
12
/l) 4.4 0.05
a
6.60 0.47
b
5.36 0.28
c
<0.0001
Thrombocytes (10
9
/l) 128 8
a
445 26
b
134 19
a
0.0037
Hemoglobin (mmol/l) 5.70 0.06
a
7.70 0.35
b
6.80 0.30
b
<0.0001
Hematocrit (l/l) 0.320 0.003
a
0.410 0.025
b
0.340 0.017
a
0.0040
Means SEM. Values in the same row not sharing the same letters are significantly different (P<0.05)
Nguyen et al. 55
(Enterobacteriaceae; Figure 5C). Principal component
analysis and analysis of similarities (ANOSIM) from
sequencing data also showed that the colonic micro-
biota in CON and ORA pigs differed significantly
(R ¼0.38, P<0.05). For those pigs with detected bac-
teria in blood and organs, Enterococcus was the pre-
dominant genus in blood, whereas both Enterococcus
and Staphylococcus were predominant groups in bone
marrow and spleen (Figure 5D). Enterobacter was not
found in blood, and only present in spleen and bone
marrow in a few pigs. Together, these data suggest that
oral antibiotics efficiently eliminated most gut Gram-
positive bacteria and thereby reduced translocation and
bacteremia.
Discussion
Colonization of the gut, skin and lungs with billions of
bacteria during the first days after birth is a challenging
event for newborns, especially preterm neonates that
show immature mucosal and systemic immune systems.
Consequently, a large proportion of preterm infants
Neutrophils
Neutrophils
Monocytes
Cell size (FSC) or granularity (SSC)
Percentage of total leukocytes (%)
Neutrophils
aa
a
a
a
c
c
c
b
b
b
b
11000
18000
180000
160000
140000
120000
150000
100000
50000
10000
Marker intensity (MFI)
Frequency (% of CD14+ cells)
Monocytes Monocytes
16000
14000
12000
10000
9000
8000
700
800
600
400
50
600
500
400
50
25
00
20
40
60
100
80
0
20
40
60
100
80
0
0
0
10
20
30
Progenitor cells %
0
CD172a
CD14
CD14high
CD14low
TLR2
a
a
a
a
a
a
a
a
a
a
b
Day 1
Day 2
Day 3
Day 1
Day 2
Day 3
Day 1
Day 2
Day 3
Day 1
Day 2
Day 3
Day 1
Day 2
Day 3
Day 1
Day 2
Day 3
Day 1
Day 2
Day 3
Day 1
Day 2
Day 3
b
b
b
b
b
b
b
b
b
b
c
FSC
SSC
c
b
b
b
c
(a) (b)
(c) (d)
Figure 2. Development of leukocyte subsets during the first 3d of life across all three groups of preterm pigs. Developmental and
maturational status of neutrophils and monocytes measured by (a, b) expression of markers CD172a, CD14 and TLR2, (c) cell size and
granularity. (d) Changes in frequency of blood progenitor cells. Values (means SEM, n¼55) across d 1–3 in the same blood cell
subsets not sharing the same letters are significantly different (P<0.05).
56 Innate Immunity 22(1)
(20–40%) experience one or more episodes of infection
and sepsis during the first few weeks after birth.
1
Systemic entry of bacteria often arises from the indwell-
ing catheters that are required to allow intensive med-
ical and nutritional treatments.
39
Systemic treatment
with broad-spectrum antibiotics is common practice
for infants born from mothers with prenatal infec-
tions,
40
or for infants with suspected NEC.
41
Gut bac-
terial overgrowth, coupled with low species diversity
and high intestinal permeability, are associated with
NEC in both infants and animal models,
28
but it
remains unclear if bacterial translocation from the
immature gut plays an important role in sepsis and
impaired immunity. We used preterm pigs as a model
for preterm infants as they are highly sensitive to NEC
and sepsis and show many of the normal signs of
physiological immaturity.
27
We described the develop-
ment of blood leukocyte subsets during the first days of
life and documented that only oral, and not systemic,
antibiotics, delayed gut colonization, reduced gut per-
meability and bacteremia, and was associated with
enhanced maturation of systemic innate immunity.
IL-6 - TLR2 agonist
IL-6 - TLR1/2 agonist
IL-6 - TLR4 agonist
TNF-α - TLR2 agonist
TNF-α - TLR1/2 agonist
TNF-α - TLR4 agonist
800
600
400
200
0
0
0
TLR2 agonist (cells/mL)
TLR1/2 agonist (ng/mL)
TLR4 agonist (ng/mL) TLR4 agonist (ng/mL)
b
b
b
b
b
a
a
a
a
a
101
10–4 10–3 10–2
10–2 10–2
10–1
10–1 10–1
102103104105
100
100100
101101
102102
0
TLR1/2 agonist (ng/mL)
10–4 10–3 10–2 10–1 100
1060
TLR2 agonist (cells/mL)
101102103104105106
IL-6 (pg/mL)
800
600
400
200
0
00
Preterm pigs Adult pi
g
s
1400
1200
1000
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200
IL-6 (pg/mL)
IL-6 (pg/mL)
400
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0
TNF-α (pg/mL)
400
300
200
100
0
TNF-α (pg/mL)
TNF-α (pg/mL)
(a) (b)
(c) (d)
(e) (f)
Figure 3. Newborn preterm whole blood demonstrated impaired TLR2-mediated cytokine production. Pro-inflammatory cytokine
responses (TNF-aand IL-6) following whole blood stimulation with (a, b) TLR2 agonist (HKLM cells), (c, d) TLR1/2 agonist
(Pam
3
CSK
4
) and (E, F) TLR4 agonist (LPS) at increasing concentrations in preterm pigs at birth (pooled blood from 20–22 pigs each
litter, n¼3 from three litters) compared with adult pigs (n¼3 sows). Values represent means SEM, and curves with increasing
concentrations of agonists not sharing the same letters are significantly different (P<0.01).
Nguyen et al. 57
Our results help to understand the important role of the
gut microbiota for the high sensitivity to sepsis and
NEC within the first few weeks after preterm birth.
First, we showed that systemic immunity was imma-
ture in newborn preterm pigs as shown by their reduced
number of blood leukocytes and higher number of pro-
genitor cells, compared with newborn term or adult
pigs. This is in line with the common knowledge that
quantitative and qualitative leukocyte deficiencies pre-
dispose preterm infants to infections, sepsis,
42,43
and
potentially also to NEC.
14–16
Regardless, the possible
direct link between NEC and systemic immunity is not
clear, neither in animal model studies nor in studies of
human preterm infants.
In human and porcine newborns, severe NEC lesions
are rare before enteral feeding is approaching full vol-
umes (>120 ml/kg/d) and NEC development is highly
diet-dependent, with infant formula being associated
with the highest NEC sensitivity. Consequently, our
observations on blood cell maturation during the first
3 d after birth with minimal enteral nutrition represent
the development during the immediate postnatal period
in the absence of NEC processes. Consistent with the
known ontogeny of systemic immunity in both humans
and pigs,
1,4,5,7
preterm pigs demonstrated increasing
postnatal maturation during d 1–3, with decreasing cell
size and CD172a expression, and increasing CD14
expression in blood neutrophils and monocytes.
However, compared with CON and SYS pigs, ORA
pigs showed enhanced neutrophil maturation, although
these effects may be indirectly induced via changes in the
gut microbiota. ORA pigs also showed limited bacterial
adherence to the intestinal mucosa (low FISH score) and
reduced bacteremia (with no bacteria detected in blood),
probably because of reduced gut bacterial density and
permeability in response to oral antibiotics. These com-
bined effects of oral antibiotics may benefit NEC resist-
ance, as well as the systemic defense against infections
after preterm birth. In contrast, less mature neutrophils,
more inflammatory monocytes (higher CD14 expres-
sion) and extensive translocation may be the systemic
consequences of a more normal bacterial colonization
in the gut of CON pigs. Parameters in SYS were inter-
mediate between those of ORA and CON, probably
indicating both a systemic and a marginal luminal
effect of systemic antibiotics to the SYS pigs.
Day 2: Neutrophil CD172a Day 3: Monocyte CD14
Day 3: Monocyte granularity
Day 3: Progenitor cells
Day 3: Neutrophil CD172a
Day 3: Neutrophil cell size
Neutrophils
(a) (b) Monocytes and progenitor cells
11000
11000
170000
160000
150000
140000
10000
10000
10000
50000
40000
30000
10000
9000
9000
8000
7000
6000
1000
8000
1000
0
0
00
10
20
30
CON SYS ORA CON SYS ORA
0
0
200
400
600
800
a
a
a
a
ab
ab
a
b
b
b
bb
P = 0.08
P = 0.05
Maker intensity (MFI)
Maker intensity (MFI)
Granularity (SSC)
Progenitor
cells (% of blood cells)
Cell size (FSC)
Maker intensity (MFI)
Figure 4. Effects of antibiotics on the development of blood leukocytes during the first 3 d of life in preterm pigs (CON, n¼17;
ORA, n¼18; SYS, n¼17). (a) neutrophils; (b) monocytes and progenitor cells. Values (means SEM) from each day not sharing
common letters are significantly different (P<0.05).
58 Innate Immunity 22(1)
The gut microbiota was dominated by Enterococcus
and Clostridium (CON pigs) or Enterobacteriacae (SYS
and ORA pigs), but not by Staphylococcus. This suggests
that a small proportion of the Staphylococcus present in
the blood and immune organs may be derived from
catheter-associated bacteremia, rather than bacterial
translocation across the leaky gut, similar to the frequent
observation of systemic infections with Staphylococcus in
human preterm infants.
39
Conversely, the high
Enterococcus load in blood, spleen and bone marrow
of CON and SYS pigs are likely gut-derived bacteria
translocated from the gut. We speculate that absence
of bacteremia in ORA pigs may be explained by the
reduced bacterial translocation from the gut due to
delayed gut colonization and low permeability. The
resulting blood neutrophil maturation may follow from
these effects and future studies using germ-free, NEC-
resistant preterm pigs would be important to confirm
these underlying mechanisms.
CD14, MD2 and TLR4 mediate detection of LPS
from Gram-negative bacteria, whereas CD14 and
TLR2 mediate signaling by bacterial lipoproteins from
Gram-positive bacteria. Expression of these receptors on
blood neutrophils and monocytes increases with gesta-
tional age from very preterm to term infants.
1,13,44
Bacteremia caused by Gram-positive bacteria is also
associated with increased mononuclear cell CD14 and
TLR2 expression.
9,45
Consistent with these observations,
we observed in the current study increased CD14 inten-
sity in preterm blood monocytes and neutrophils with
advancing age. However, TLR2 intensity was low and
did not change over time on neutrophils and monocytes
from any of the three groups, even though Gram-
positive bacteria dominated in the gut lumen of CON
pigs and also the blood in CON and SYS pigs. The low
TLR2 expression may explain the impaired TLR2-
mediated cytokine production in preterm blood. In the
preterm pig intestine, TLR2 gene expression, but not
TLR4, increases following enteral feeding.
46,47
This is
in line with our data showing a predominance of
Gram-positive bacteria in the gut of CON pigs, as this
may trigger the development of intestinal TLR2 expres-
sion. This does not appear to induce a parallel increase
in blood leukocyte TLR2 expression and suggest a defi-
cient systemic immunity to Gram-positive bacteria in
preterm pigs during the first few days of life. In contrast,
TLR4-mediated preterm blood cytokine responses were
more pronounced and Gram-negative bacteria were neg-
ligible in blood of all three groups of pigs. This suggests
that a relatively mature blood leukocyte response to
Gram-negative bacteria reduces bacteremia caused by
these pathogens in newborn preterm pigs. ORA pigs
showed no evidence of bacteremia, implying that oral
broad-spectrum antibiotics may prevent Gram-positive
bacteremia in preterm neonates, partly via a reduced gut
permeability, bacterial colonization and translocation.
Bacterial load in intestine Bacterial load in blood, bone marrow and spleen
Predominant genera in blood,
bone marrow and spleen
Bacterial abundance (%)
Bacterial load (CFU/mL
blood or CFU/g tissue)
Bacterial load (CFU/mL
blood or CFU/g tissue)
Predominant genera in intestine
00
0
30
60
500
1000
1500
2000
3000
2500
100
200
2000
4000
6000
8000
10000
0
20
40
60
80
100
aa
b
b
Blood SpleenBone marrow
Blood Spleen
Bone marrow
b
a
a
a
b
CON
SYS
ORA
CON SYS ORA
CON
Enterococcus spp. Enterobacteriaceae/unclassified
Enterobacteriaceae/unidentified
Clostridium spp.
Staphylococcus spp.
Enterococcus spp.
Enterobacter spp.
Staphylococcus spp.
Others
SYS ORA
2
Log10 of 16S rRNA copies
4
6
8
(a) (b)
(c) (d)
Figure 5. Microbiology. (a, b) Total bacterial load and (c, d) predominant genera (c-d) in the gut (n¼7–13/group), and in blood,
spleen and bone marrow (n¼16–17/group). Values (means SEM) for bacterial load in gut, blood, bone marrow or spleen not sharing
common letters are significantly different (P<0.05).
Nguyen et al. 59
Day 12345
CON pigs
SYS pigs
ORA pigs
Systemic
antibiotics
Oral
antibiotics
a
a
a
b
c
c
d
d
e
e
f
f
Gram-positive bacteria
Gram-negative bacteria
Intestinal epithelial cells
TLR2
Gut lumen
Mucus layer
Epithelial laye
r
Blood stream
Neutrophils
Monocytes
Lymphocytes
CD172a
Necrotic intestinal
epithelial cells
Figure 6. Summary of the suggested mechanisms that relate to neonatal antibiotics treatment of preterm pigs. The diagram illus-
trates blood leukocyte development, gut colonization, translocation and NEC development (CON, SYS and ORA pigs) during the first
5 d after preterm birth. (a) Gut colonization occurs rapidly after birth with a high load of Gram-positive bacteria in CON pigs and
Gram-negative bacteria in SYS pigs, whereas colonization is delayed with few bacteria in ORA pigs (b). Oral antibiotics delay
colonization and (c) enhance blood neutrophil maturation, as reflected by reduced cell size and CD172a expression on d 2–3. High
bacterial load in the gut of CON and SYS pigs facilitates (c) Gram-positive bacterial adherence to the intestinal mucosa and (d)
translocation of bacteria and bacterial products into the blood stream. The combined effects of high bacterial load, translocation and
less neutrophil maturation contribute to NEC development in CON and SYS pigs with (e) damage to epithelial cells, (f) leukocyte
infiltration and increased intestinal permeability.
60 Innate Immunity 22(1)
With regard to NEC prevention, oral was superior to
systemic antibiotic treatment, probably because only
oral treatment depressed the rapid gut bacterial colon-
ization and reduced gut permeability. Conversely, a high
gut bacterial load, combined with an immature systemic
innate immune response against Gram-positive bacteria,
may predispose to bacteremia and NEC. More inflam-
matory monocytes with high CD14 expression on d 3
and increased monocyte counts on d 5, as shown in
CON pigs, may also play a role in NEC pathogenesis.
This is in line with the observed higher monocyte CD14
expression and monocyte granularity in NEC pigs.
When the bacterial translocation occurs in SYS pigs,
systemic antibiotics may help to clear the blood from
intact bacteria but not from the products released from
dead bacteria. These may then contribute to the systemic
inflammation often associated with NEC.
The suggested mechanisms based on our study of
neonatal antibiotics to preterm pigs are summarized
in Figure 6. We demonstrate that oral administration
of antibiotics during the first few days after preterm
birth enhances maturation of blood neutrophils,
diminishes gut colonization and permeability, and pre-
vents bacterial translocation. These combined effects
may appear to protect formula-fed preterm neonates
against bacteremia and NEC-like lesions in the period
immediately after birth. In contrast, control formula-
fed neonates are associated with a higher gut bacterial
load, increased blood monocyte CD14 expression,
increased bacterial translocation and inflammation,
damage of epithelial cells and greater sensitivity to
NEC (Figure 6). Future investigations comparing pre-
term and term pigs at different time points after birth
and fed different enteral diets are important to further
elucidate the mechanisms of immune maturation in
pigs, used as models for human preterm and term
infants. A very high sensitivity to both NEC and
sepsis in preterm pigs indicate that the gut and
immune systems may be even more immature in pre-
term pigs vs. infants,
27
but this also makes pigs very
sensitive models to study factors that influence the
early maturation of these systems.
Our study illustrates important principles for the post-
natal development of gut bacterial colonization, systemic
immunity and NEC sensitivity in response to systemic or
oral antibiotics to formula-fed preterm neonates. While
concerns about increased microbial resistance and
adverse long-term effects may prevent widespread use
of neonatal short-term antibiotics, even for preterm new-
borns, it remains important to investigate novel prevent-
ive and therapeutic regimens for these sensitive pediatric
patients. Antimicrobial proteins and peptides in natural
milk may mimic the effects of oral antibiotics and there is
a need to understand the combined effects of early milk
and microbiota on the developing gut and immune sys-
tems in order to support health of preterm infants in both
the short and long term.
Acknowledgements
We thank Elin Skytte and Kristina Møller for animal proced-
ures, and Malene S Cilieborg for assistance in FISH analysis.
Ms Kristin Johnson, a medical graphics specialist at Boston
Children’s Hospital, is acknowledged for assistance with
Figure 6.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with
respect to the research, authorship, and/or publication of this
article.
Funding
The author(s) disclosed receipt of the following financial sup-
port for the research, authorship, and/or publication of this
article: This work was supported by the Danish Strategic
Research Council (NEOMUNE, grant number 12-132401).
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