Maternal Tn Immunization Attenuates Hyperoxia-Induced Lung Injury in Neonatal Rats Through Suppression of Oxidative Stress and Inflammation

Abstract

Hyperoxia therapy is often required to treat newborns with respiratory disorders. Prolonged hyperoxia exposure increases oxidative stress and arrests alveolar development in newborn rats. Tn antigen is N-acetylgalactosamine residue that is one of the most remarkable tumor-associated carbohydrate antigens. Tn immunization increases the serum anti-Tn antibody titers and attenuates hyperoxia-induced lung injury in adult mice. We hypothesized that maternal Tn immunizations would attenuate hyperoxia-induced lung injury through the suppression of oxidative stress in neonatal rats. Female Sprague–Dawley rats (6 weeks old) were intraperitoneally immunized five times with Tn (50 μg/dose) or carrier protein at biweekly intervals on 8, 6, 4, 2, and 0 weeks before the day of delivery. The pups were reared in room air (RA) or 2 weeks of 85% O2, creating the four study groups: carrier protein + RA, Tn vaccine + RA, carrier protein + O2, and Tn vaccine + O2. The lungs were excised for oxidative stress, cytokine, vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) expression, and histological analysis on postnatal day 14. Blood was withdrawn from dams and rat pups to check anti-Tn antibody using western blot. We observed that neonatal hyperoxia exposure reduced the body weight, increased 8-hydroxy-2-deoxyguanosine (8-OHdG) expression and lung cytokine (interleukin-4), increased mean linear intercept (MLI) values, and decreased vascular density and VEGF and PDGF-B expressions. By contrast, Tn immunization increased maternal and neonatal serum anti-Tn antibody titers on postnatal day 14, reduced MLI, and increased vascular density and VEGF and PDGF-B expressions to normoxic levels. Furthermore, the alleviation of lung injury was accompanied by a reduction in lung cytokine and 8-OHdG expression. Therefore, we propose that maternal Tn immunization attenuates hyperoxia-induced lung injury in neonatal rats through the suppression of oxidative stress and inflammation.

Full text

ORIGINAL RESEARCH
published: 04 April 2019
doi: 10.3389/fimmu.2019.00681
Frontiers in Immunology | www.frontiersin.org 1April 2019 | Volume 10 | Article 681
Edited by:
Pedro A. Reche,
Complutense University of Madrid,
Spain
Reviewed by:
Silvia Sánchez-Ramón,
Complutense University of Madrid,
Spain
Javier Carbone,
Hospital General Universitario
Gregorio Marañón, Spain
Francisco Javier Cubero,
Complutense University of Madrid,
Spain
*Correspondence:
Chung-Ming Chen
cmchen@tmu.edu.tw
Specialty section:
This article was submitted to
Vaccines and Molecular Therapeutics,
a section of the journal
Frontiers in Immunology
Received: 27 December 2018
Accepted: 12 March 2019
Published: 04 April 2019
Citation:
Chen C-M, Hwang J and Chou H-C
(2019) Maternal Tn Immunization
Attenuates Hyperoxia-Induced Lung
Injury in Neonatal Rats Through
Suppression of Oxidative Stress and
Inflammation. Front. Immunol. 10:681.
doi: 10.3389/fimmu.2019.00681
Maternal Tn Immunization
Attenuates Hyperoxia-Induced Lung
Injury in Neonatal Rats Through
Suppression of Oxidative Stress and
Inflammation
Chung-Ming Chen 1,2
*, Jaulang Hwang 3and Hsiu-Chu Chou 4
1Department of Pediatrics, Taipei Medical University Hospital, Taipei, Taiwan, 2Department of Pediatrics, School of Medicine,
College of Medicine, Taipei Medical University, Taipei, Taiwan, 3Taipei Cancer Center, Taipei Medical University, Taipei, Taiwan,
4Department of Anatomy and Cell Biology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan
Hyperoxia therapy is often required to treat newborns with respiratory disorders.
Prolonged hyperoxia exposure increases oxidative stress and arrests alveolar
development in newborn rats. Tn antigen is N-acetylgalactosamine residue that is
one of the most remarkable tumor-associated carbohydrate antigens. Tn immunization
increases the serum anti-Tn antibody titers and attenuates hyperoxia-induced lung
injury in adult mice. We hypothesized that maternal Tn immunizations would attenuate
hyperoxia-induced lung injury through the suppression of oxidative stress in neonatal
rats. Female Sprague–Dawley rats (6 weeks old) were intraperitoneally immunized five
times with Tn (50 µg/dose) or carrier protein at biweekly intervals on 8, 6, 4, 2, and 0
weeks before the day of delivery. The pups were reared in room air (RA) or 2 weeks
of 85% O2, creating the four study groups: carrier protein +RA, Tn vaccine +RA,
carrier protein +O2, and Tn vaccine +O2. The lungs were excised for oxidative
stress, cytokine, vascular endothelial growth factor (VEGF) and platelet-derived growth
factor (PDGF) expression, and histological analysis on postnatal day 14. Blood was
withdrawn from dams and rat pups to check anti-Tn antibody using western blot.
We observed that neonatal hyperoxia exposure reduced the body weight, increased
8-hydroxy-2-deoxyguanosine (8-OHdG) expression and lung cytokine (interleukin-4),
increased mean linear intercept (MLI) values, and decreased vascular density and
VEGF and PDGF-B expressions. By contrast, Tn immunization increased maternal and
neonatal serum anti-Tn antibody titers on postnatal day 14, reduced MLI, and increased
vascular density and VEGF and PDGF-B expressions to normoxic levels. Furthermore,
the alleviation of lung injury was accompanied by a reduction in lung cytokine and
8-OHdG expression. Therefore, we propose that maternal Tn immunization attenuates
hyperoxia-induced lung injury in neonatal rats through the suppression of oxidative stress
and inflammation.
Keywords: hyperoxia, vaccine, interleukin-4, 8-hydroxy-2′-deoxyguanosine, mean linear intercept,
von Willebrand factor

Chen et al. Immunization and Hyperoxia-Induced Lung Injury
INTRODUCTION
Respiratory distress syndrome is a major cause of morbidity
and mortality in preterm neonates (1). Hyperoxia therapy is
often required to treat newborns with respiratory disorders.
However, supplemental oxygen administered to newborn infants
with respiratory failure increases oxidant stress and leads to
lung injury. The rat model is appropriate to study the effects of
hyperoxia on preterm infants with respiratory distress because
rats are born at the saccular stage, equivalent to an ∼30 week
human gestation (2). Prolonged exposure of neonatal rodents to
hyperoxia resulted in decreased alveolar septation and increased
terminal air space size, similar to human bronchopulmonary
dysplasia (3,4). Despite early surfactant therapy, optimal
ventilation strategies, and increased use of noninvasive positive
pressure ventilation, bronchopulmonary dysplasia remains a
major cause of morbidity and mortality during the first year
of life, and many infants experience significant respiratory
morbidity, including decreased response to acute hypoxia,
increased airway reactivity, and development of obstructive
airway disease throughout childhood (5–7). No effective clinical
therapy is currently available to prevent the development and
long-term pulmonary sequelae of bronchopulmonary dysplasia.
Tn antigen is N-acetylgalactosamine residue that is α-
linked to a serine or threonine residue, which is one of
the most remarkable tumor-associated carbohydrate antigens,
often offered to mucin-type carbohydrates (8). Studies have
demonstrated that inflammatory cytokines can promote glycan
epitope by regulating specific glycosyltransferases (9,10). Using
the linear array epitope technology, Chiang et al. developed
an anti-Tn vaccine that induces anti-Tn antibodies with high
specificity and high affinity in mice (11). These results suggest
that Tn may show immunogenicity and protection in preclinical
animal studies. Tn immunization attenuates hyperoxia-induced
lung injury in adult mice by inhibiting the nuclear factor-kappa
B (NF-κB) activity (12). The effects of Tn immunization on
neonatal hyperoxia-induced lung injury are unknown. Therefore,
we hypothesize that the maternal Tn immunizations would
attenuate hyperoxia-induced lung injury in neonatal rats. This
study investigated the protective effects and mechanisms of
Tn immunization on lung inflammation and development in
neonatal rats exposed to hyperoxia.
MATERIALS AND METHODS
Tn Vaccine Preparation
Tn vaccine was prepared by conjugating Tn to a novel carrier
protein as described previously (11). Tn was conjugated to
mFc(Cys42)Histag2 or GST(Cys6)Histag2 at a glycotope–carrier
protein weight ratio of 5:1. The conjugation was performed
in a buffer containing 20 mM sodium phosphate, pH 7.9,
8 M urea, 500 mM imidazole, and 0.2 mM tris(2-carboxyethyl)
phosphine (TCEP). After 48 h, the conjugate was refolded in
phosphate-buffered saline (PBS) with 0.2 mM TCEP. GST(Cys6)
was dialyzed against PBS with 0.2 mM TCEP. Different
glycotopes and a linker (N-succinimidyl-6-maleimidocaproate)
were conjugated to GST(Cys6) at 4◦C for 48 h.
Animal Model and Experimental Groups
Female Sprague–Dawley rats (6 weeks old) were obtained from
BioLASCO Taiwan Co., Ltd and were housed in individual
cages with 12-h light–dark cycles. Laboratory food and water
were available ad libitum. The female rats were randomly
assigned to the Tn immunization or control treatment groups
(Supplement Figure 1). The Tn immunization strategy consisted
of an intraperitoneal injection of Tn (50 µg/dose) in 0.5 mL
normal saline, and the control immunization consisted of the
intraperitoneal injection of the same volume of carrier protein.
The immunizations were administered five times at biweekly
intervals on 8, 6, 4, 2, and 0 weeks before the day of delivery.
Female rats in estrus or proestrus were placed in cages with adult
male rats (two females for each male) for 12 h. The following
morning, mating was confirmed by the presence of a vaginal
plug, which was considered day 0 of gestation. The dams were
allowed to deliver vaginally at term. Within 12 h of birth, litters
were pooled and randomly redistributed to the newly delivered
mothers, and the pups were then randomly assigned to room air
(RA) or oxygen-enriched atmosphere (O2) treatment. The pups
in the O2treatment subgroups were reared in an atmosphere
containing 85% O2from postnatal days 1 to 14. The pups in the
control subgroups were reared in normobaric RA for 14 days.
Four study groups were obtained as follows: carrier protein +
RA, Tn vaccine +RA, carrier protein +O2, and Tn vaccine
+O2. To avoid oxygen toxicity in the nursing mothers, they
were rotated between the O2treatment and RA control litters
every 24 h. An oxygen-rich atmosphere was maintained in a
transparent 40 ×50 ×60-cm plexiglass chamber receiving
O2continuously at 4 L/min. Oxygen levels were monitored
using a ProOx P110 monitor (NexBiOxy, Hsinchu, Taiwan). On
postnatal day 14, pups from each group were deeply anesthetized
with an overdose of isoflurane, and body and lung weights were
noted. Blood was withdrawn from dams and rat pups to check
anti-Tn antibody using western blot. The study protocol was
approved by the Institutional Animal Care and Use Committee
of Taipei Medical University.
Western Blot Analysis of the Serum
Anti-Tn Antibody
Solubilized proteins were separated using SDS-PAGE and
were electrophoretically transferred to polyvinylidene difluoride
(PVDF) membranes. PVDF membranes were rinsed in TBS
buffer and blocked for 1 h with TBS buffer containing 5% skim
milk. After washing with TBST (Tris-buffered saline, 0.1% Tween
20), the PVDF membranes were incubated overnight with rat
serum (1:1000) dissolved in antibody buffer. After multiple
washing with TBST, the membranes were incubated for 45 min
with Jackson AffiniPure Donkey AntiRat IgG (H+L) (1:5000,
Jackson ImmunoResearch Laboratories, Inc., West Grove, PA,
USA). Membranes were washed, and immunoreactive proteins
were detected using Immun-Star assay kit (Bio-Rad) following
the manufacturer’s suggestions.
Lung Histology
To standardize analysis, sections were obtained from the right
middle lobe of the right lung. The lung tissue was immersed
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Chen et al. Immunization and Hyperoxia-Induced Lung Injury
with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4)
at 4◦C for 24 h. The tissues were then dehydrated in alcohol,
cleared in xylene, and embedded in paraffin. Five-micrometer
sections were stained with hematoxylin and eosin, examined
using light microscopy, and assessed for lung morphometry.
Mean linear intercept (MLI), an indicator of mean alveolar
diameter, was assessed in 10 nonoverlapping fields (13). Vascular
density was determined with von Willebrand factor (vWF)
immunohistochemistry reaction (see below).
Immunohistochemistry of
8-Hydroxy-2′-Deoxyguanosine, von
Willebrand Factor, Vascular Endothelial
Growth Factor, Platelet-Derived Growth
Factor-B, Inducible Nitric Oxide Synthase,
and YM-1
Immunohistochemical staining was performed on 5-µm paraffin
sections using immunoperoxidase visualization. After routine
deparaffinization, heat-induced epitope retrieval was performed
by immersing the slides in 0.01 M sodium citrate buffer
(pH 6.0). To block the endogenous peroxidase activity and
nonspecific binding of antibodies, the sections were preincubated
for 1 h at room temperature in 0.1 M PBS containing 10%
normal goat serum and 0.3% H2O2. The sections were then
incubated for 20 h at 4◦C with mouse monoclonal anti-8-
hydroxy-2′-deoxyguanosine (8-OHdG) antibody (1:100; Abcam
Inc., Cambridge, MA, USA), rabbit polyclonal antivWF antibody
(1:100; Abcam), rabbit polyclonal antivascular endothelial
growth factor (VEGF) antibody (1:50; Santa Cruz Biotechnology,
Inc., CA, USA), rabbit polyclonal antiplatelet-derived growth
factor (PDGF)-B antibody (1:50; Santa Cruz Biotechnology, Inc.),
rabbit polyclonal anti-inducible nitric oxide synthase (iNOS)
(1:100; Thermo Fisher Scientific, Rackford, IL, USA), or rabbit
polyclonal anti-Ym-1 (1:25; STEMCELL Technologies Inc.,
Vancouver, Canada) as primary antibodies. The sections were
then treated for 1 h at 37◦C with biotinylated goat anti-mouse
or rabbit IgG (1:200, Jackson ImmunoResesarch Laboratories
Inc., PA, USA). Following the reaction produced using reagents
from an avidin–biotin complex kit (Vector Laboratories, Inc.,
CA, USA), the reaction products were visualized using a
diaminobenzidine substrate kit (Vector Laboratories, Inc.)
according to the recommendations of the manufacturer. The
8-OHdG staining was quantified by considering the positively
stained nuclei per high-power field. Positively stained cells
were counted in five fields randomly selected from each
section using a light microscope (magnification: ×400), and
results were expressed as the percentage of positively stained
nuclei to total cells. Microvessel density was determined by
counting the vessels with the positive vWF stained in an
unbiased manner and a minimum of four random lung fields
at ×400 magnifications (14). The automatic object counting and
measuring process was used to quantify the immunoreactivity in
the sections. This generated a percentage of positively stained
cells, and the values were expressed as a labeling index (%).
The positive immunostaining of lung parenchyma for iNOS and
Ym-1 were measured at ×400 magnification by the density of
immunostained chromogen (0.1 mm2) using the Image Pro Plus
(Media Cybernetics, Silver Spring, USA).
Cytokine Assay
Approximately 100 mg of lung tissue from each pup was
homogenized, sonicated, and centrifuged at 500 ×g for 20 min
at 4◦C to remove cellular debris according to the manufacturer’s
instructions. The levels of interleukin-4 (IL-4) in the supernatants
were determined using the enzyme-linked immunosorbent assay
kit (MyBioSource, San Diego, CA, USA) as IL-4 expression
was significantly increased in the lungs from hyperoxia-exposed
neonatal rats and mice (15,16).
Western Blot Analysis of Growth Factors
Lung tissues were homogenized in ice-cold buffer containing
50 mM Tris·HCl (pH 7.5), 1 mM EGTA, 1 mM EDTA, and
protease inhibitor cocktail (complete minitablets; Roche,
Mannheim, Germany). The samples were sonicated and then
centrifuged at 500 gfor 20 min at 4◦C to remove cellular debris.
Proteins (30 µg) were resolved on 12% SDS-PAGE under
reducing conditions and electroblotted to a PVDF membrane
(ImmobilonP, Millipore, Bedford, MA, USA). After blocking
with 5% nonfat dry milk, the membranes were incubated with
antibody against VEGF (1:1000; Santa Cruz Biotechnology,
Inc.), PDGF-B (1:1000; Santa Cruz Biotechnology, Inc.), or
anti-β-actin (1:20,000; Sigma-Aldrich, St. Louis, MO, USA)
and subsequently with horseradish peroxidase-conjugated
goat anti-rabbit IgG or anti-mouse IgG (Pierce Biotechnology,
Rockford, USA). Protein bands were detected using SuperSignal
Substrate from Pierce. Densitometric analysis was performed
to measure the intensity of VEGF, PDGF-B, and β-actin bands
using AIDA software.
Statistical Analysis
All data were presented as mean ±SD. Statistical analyses
were performed using a two-way analysis of variance with a
Bonferroni post hoc test for multiple group comparisons. The
survival rate was evaluated using the Kaplan–Meier method, and
log-rank test was used for intergroup comparisons. Differences
were considered statistically significant when p<0.05.
RESULTS
Three Tn immunization-treated and three carrier protein-treated
female rats were successfully mated with male rats. Six dams
gave birth to a total of 46 pups; 23 pups each were randomly
distributed to the RA and hyperoxia groups. A total of 11 and
12 pups received carrier protein and Tn immunization in the
RA groups, and 11 and 12 pups received carrier protein and Tn
immunization in the hyperoxia groups.
Western Blot Analysis of Serum
Anti-Tn Antibody
PE-(PC7)Tn recognized predominantly one major band
(anti-Tn antibody), which was not recognized by PE-(PC7)
(Figure 1). Mothers and pups receiving Tn immunization
exhibited a dense anti-Tn antibody band, whereas mothers
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Chen et al. Immunization and Hyperoxia-Induced Lung Injury
FIGURE 1 | Western blot analysis of serum anti-Tn antibody in dams and rat pups on postnatal day 14. PE-(PC7)Tn predominantly recognized one major band
(anti-Tn antibody), which was not recognized by PE-(PC7). Mothers and pups who received Tn immunization exhibited a dense anti-Tn antibody band, whereas
mothers and pups who received carrier protein immunization did not exhibit anti-Tn antibody. The diagram illustrates representative data from three experiments.
and pups receiving carrier protein immunization did not
exhibit anti-Tn antibody.
Survival
The rats reared in the carrier protein +RA or Tn vaccine
+RA group all survived (Figure 2). The rats reared in
the carrier protein +O2or Tn vaccine +O2group
exhibited a lower survival rate after postnatal day 7.
On postnatal day 14, the survival rate between the rats
treated with the carrier protein or Tn immunization
were comparable.
Body Weight, Lung Weight, and
Lung-To-Body Weight Ratios
The rats in the carrier protein +O2or Tn vaccine +O2
group exhibited significantly lower body and lung weights
on postnatal day 14 than those reared in the carrier protein
+RA or Tn vaccine +RA group (Table 1). Maternal
Tn immunization increased the body weight on postnatal
day 14 in rats reared in RA or hyperoxia. The rats in
the carrier protein +O2group exhibited a significantly
higher lung-to-body weight ratio than those in the other
three groups.
Immunohistochemistry for 8-OHdG
To investigate whether maternal Tn immunization reduced
oxidative stress in neonatal hyperoxia-exposed rat lungs, we
used immunohistochemical assays for oxidative stress marker 8-
OHdG. The 8-OHdG immunoreactivity was primarily detected
in the epithelial cells (Figure 3A). The rats in the carrier protein
+O2group exhibited significantly higher number of positive 8-
OHdG cells than those in the carrier protein +RA or Tn vaccine
TABLE 1 | Body weights, lung weights, and lung-to-body weight ratios of rat
pups on postnatal day 14.
Treatment nBody weight (g) Lung weight (g) Lung-to-
body weight
ratio (%)
Carrier protein +RA 11 20.36 ±1.43 0.33 ±0.02 1.60 ±0.12
Carrier protein +O29 15.11 ±1.58a0.31 ±0.05 2.08 ±0.48c
Tn vaccine +RA 12 23.67 ±2.12 0.36 ±0.02 1.55 ±0.11
Tn vaccine +O210 19.35 ±0.82a,b 0.33 ±0.02 1.71 ±0.11
Values are mean ±SD.
ap<0.001 vs. carrier protein +RA and Tn vaccine +RA.
bp<0.001 vs. carrier protein +O2.
cp<0.05 vs. carrier protein +RA, Tn vaccine +RA, and Tn vaccine +O2.
+RA group. Maternal Tn immunization significantly decreased
the neonatal hyperoxia-induced increase in the number of
positive 8-OHdG cells (Figure 3B).
Cytokine Level
The rats in the carrier protein +O2group exhibited
a significantly higher lung IL-4 level than those in
the carrier protein +RA or Tn vaccine +RA group
(Figure 3C). Maternal Tn immunization significantly
decreased the neonatal hyperoxia-induced increase in the lung
IL-4 levels.
Histology Results
Representative lung sections stained with hematoxylin and
eosin and vWF from maternal Tn immunization and postnatal
hyperoxia-exposed rats on postnatal day 14 are shown in
Figures 4A,B, respectively. The rats in the carrier protein +
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Chen et al. Immunization and Hyperoxia-Induced Lung Injury
FIGURE 2 | Effects of Tn immunization on the survival rate on postnatal day 14. The rats in the carrier protein +RA or Tn vaccine +RA group all survived. The rats in
the carrier protein +O2or Tn vaccine +O2group exhibited a lower survival rate after postnatal day 7. On postnatal day 14, the survival rate between the rats treated
with carrier protein or Tn immunization were comparable.
FIGURE 3 | (A) Representative immunohistochemistry of 8-hydroxy-2′-deoxyguanosine (8-OHdG), (B) quantitative analysis of 8-OHdG-positive cells, and (C) lung
IL-4 in 14-day-old rats in the carrier protein +RA, Tn vaccine +RA, carrier protein +O2, or Tn vaccine +O2group. Positive staining is indicated in brown (arrow).
The rats in the carrier protein +O2group exhibited a significantly higher number of positive 8-OHdG cells and IL-4 levels than those in the carrier protein +RA or Tn
vaccine +RA group. Maternal Tn immunization significantly decreased the hyperoxia-induced increase in the number of positive 8-OHdG cells and IL-4 levels. *p<
0.05, ***p<0.001.
O2group exhibited a significantly higher MLI and lower
vascular density than those in the carrier protein +RA or Tn
vaccine +RA group. Maternal Tn immunization improved the
hyperoxia-induced alteration in the MLI and vascular density to
normoxic levels.
Immunohistochemistry and Western
Blotting of VEGF and PDGF-B
Representative immunohistochemistry of VEGF and PDGF-B
are shown in Figures 5A,B, respectively. The VEGF and PDGF-B
immunoreactivities were primarily detected in the endothelial
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Chen et al. Immunization and Hyperoxia-Induced Lung Injury
FIGURE 4 | Representative H&E stained lung sections for histology observation and (A) mean linear intercept assessment and (B) immunohistochemistry of vWF and
semiquantitative analysis for vascular density in lung of 14-day-old rats in the carrier protein +RA, Tn vaccine +RA, carrier protein +O2, or Tn vaccine +O2group.
The rats in the carrier protein +O2group exhibited a significantly higher MLI and lower vascular density than those in the carrier protein +RA or Tn vaccine +RA
group. Maternal Tn immunization reversed the MLI and vascular density to normoxic levels. **p<0.01, ***p<0.001.
and epithelial cells. The rats in the carrier protein +O2
group exhibited significantly lower VEGF and PDGF-B protein
expression than those in the carrier protein +RA or Tn vaccine
+RA group. Maternal Tn immunization significantly increased
the hyperoxia-induced decrease in the VEGF and PDGF-B
protein expression.
M1/M2 Phenotype in Macrophages
Representative lung sections stained with iNOS (M1 macrophage
maker) and Ym1 (M2 macrophage maker) from maternal Tn
immunization and postnatal hyperoxia-exposed rats on postnatal
day 14 are shown in Figures 6A,B, respectively. The rats in the
carrier protein +O2group exhibited a significantly higher M1
phenotype macrophages and lower M2 phenotype macrophages
than those in the carrier protein +RA or Tn vaccine +
RA group (Figures 6C,D). Maternal Tn immunization reversed
the hyperoxia-induced M1/M2 macrophage polarization to
normoxia levels.
DISCUSSION
Our in vivo model revealed that maternal Tn immunization
increased maternal and neonatal serum antibody titers and
attenuated hyperoxia-induced lung injury in newborn rats, as
evidenced by reversing hyperoxia-induced increase in MLI and
decrease in vascular density and growth factors. The alleviation
of lung injury was associated with a reduction in cytokine
and 8-OHdG expression. Therefore, we proposed that maternal
Tn immunization attenuates hyperoxia-induced lung injury
in neonatal rats through the suppression of oxidative stress
and inflammation.
Tn antigen is a pan-carcinoma antigen, expressed on breast,
pancreas, colon, lung, and bladder carcinomas, being less
common in hematological malignancies (17,18). Tn is associated
with immune disorders in addition to cancers. Tn antigen can
be detected on chronic inflammatory tissues in patients with
rheumatoid arthritis and osteoarthritis (19). Tn can induce
tumor-specific IgG antibodies in mice and in nonhuman
primates under appropriate conditions (20). These findings
revealed that Tn might be an essential component in the
design of humoral-mediated vaccines and suggested that Tn
may show immunogenicity and protection in preclinical animal
studies. Tn immunization increased serum anti-Tn antibody
titers and protected against hyperoxia-induced lung injury in
adult mice through the inhibition of NF-κB activity (12).
Therefore, maternal Tn immunization may attenuate hyperoxia-
induced lung injury in neonatal mice through suppression
of inflammation.
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Chen et al. Immunization and Hyperoxia-Induced Lung Injury
FIGURE 5 | Representative immunohistochemistry and representative western blots and quantitative data for (A) VEGF and (B) PDGF-B protein expression in
14-day-old rats in the carrier protein +RA, Tn vaccine +RA, carrier protein +O2, or Tn vaccine +O2group. The rats in the carrier protein +O2group exhibited
significantly lower vascular density and VEGF and PFGF-B expression than those in the carrier protein +RA or Tn vaccine +RA group. Maternal Tn immunization in
hyperoxia-exposed rats improved vascular density and VEGF and PDGF-B expression to normoxic levels. *p<0.05, **p<0.01.
Maternal immunization provides protection to the newborns
through the transfer of vaccine-induced IgG across the placenta.
IgG is the only antibody class that significantly crosses the human
placenta. Coder et al. maternally administered radiolabeled
humanized IgG2 and found humanized IgG2 in rat embryo/fetal
tissues as early as gestation day 11 with a >1,000-fold increase
in the amount of total IgG2 by gestation day 21 (21). The
concentration of IgG2 in rat embryo/fetal tissues generally
remained unchanged from gestation day 11–17 with a slight
increase from day 17–21. Moffat et al. compared IgG2X
embryonic exposure in rats and found that fetal IgG2X plasma
concentrations increased more than six-fold from gestation
days 16–21 (22). In this study, we immunized the female
rats three times before gestation, on gestation day 7, and
at delivery and observed that Tn immunization increased
serum anti-Tn antibody titers and protected against hyperoxia-
induced lung injury in neonatal rats. These results indicate
that maternal immunization is a potential strategy to prevent
and treat neonatal diseases. Further studies are needed to
determine total IgG and complement factors to elucidate the
immunological mechanisms that mediate the beneficial effects of
maternal immunization.
Hyperoxia exposure for 7 days increased oxidative stress
in the neonatal murine lungs (23,24). 8-OHdG is a DNA
base-modified product generated by reactive oxygen species as
a marker of oxidative DNA damage and its levels in target
tissues are correlated with other oxidative stress markers (25–
27). The expression of 8-OHdG reflects the oxidative stress
level in the lung tissues and its expression was elevated in the
hyperoxia-exposed neonatal rat lung tissue and primary cultured
neonatal rat alveolar epithelial type II cells compared with the
normoxic controls (28). Positive signals for 8-OHdG increased
in the hyperoxia-exposed rats, and signals were mainly found
in the nuclei of epithelial cells. Maternal Tn immunization
significantly decreased the hyperoxia-induced increase in the
number of positive 8-OHdG cells. These results suggest that anti-
Tn antibody-suppressed oxidative stress formation and support
that anti-oxidant enzymes are effective in reducing hyperoxia-
induced neonatal lung injury (24,29).
VEGF is a potent endothelial cell mitogen that regulates
angiogenesis and alveolar development (30). PDGF is crucial
for alveolarization of the normally developing lung (31). We
determined VEGF and PDGF-B expression as their mRNA
and protein expression was decreased in the hyperoxia-exposed
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Chen et al. Immunization and Hyperoxia-Induced Lung Injury
FIGURE 6 | (A,B) Representative immunohistochemistry and (C,D) quantitative analysis of iNOS and Ym1 positive cells in 14-day-old rats in the carrier protein +RA,
Tn vaccine +RA, carrier protein +O2, or Tn vaccine +O2group. The rats in the carrier protein +O2group exhibited a significantly higher M1 phenotype (iNOS)
macrophages (arrow) and lower M2 phenotype (Ym-1) macrophages than those in the carrier protein +RA or Tn vaccine +RA group. Maternal Tn immunization
reversed the hyperoxia-induced M1/M2 macrophage polarization to normoxia levels. **p<0.01, ***p<0.001.
neonatal mice and piglet lungs (32,33). In this study, we
demonstrated that rats in the carrier protein +O2group
exhibited significantly decreased VEGF and PDGF-B expression
than those in the carrier protein +RA or Tn vaccine +RA group.
Maternal Tn immunization significantly reversed the hyperoxia-
induced decrease in VEGF and PDGF-B to normoxic levels.
These results suggest that maternal Tn immunization enhanced
vascular and alveolar development through the induction of
growth factors in neonatal rats.
In addition to the traditional host defense, inflammation,
and scavenging functions, macrophages have broader
functions, including vital roles in tissue repair and organ
development (34–36). Animal studies have demonstrated
that neonatal hyperoxia exposure increases macrophage
infiltration into alveolar airspaces (37,38). In this study, we used
immunohistochemistry to detect the macrophage infiltration
on the lung tissue sections. Macrophage phenotype was
assessed through immunostaining for iNOS (M1 macrophage
marker) and Ym1 (M2 macrophage marker) as hyperoxia
increases iNOS expression in macrophages and hyperoxia-
exposed murine lungs and inhibits the M2 phenotype in
macrophages (39,40). The rats in the carrier protein +O2
group significantly exhibited more M1 macrophages than those
in the carrier protein +RA or Tn vaccine +RA group. These
results indicate that hyperoxia promotes the M1 phenotype
in macrophages and suggest that the M1/M2 polarization
mediates the pulmonary effects of hyperoxia in the developing
lung. Our study increases the understanding of the role of
macrophage polarization in hyperoxia-induced injury to the
developing lungs.
In conclusion, we observed that Tn immunization increases
serum anti-Tn antibody titers in mothers and neonates, inhibits
lung inflammation and oxidative stress, and enhances lung
development in the neonatal hyperoxia-exposed rats. These
findings indicate that Tn activation may be involved in the
mechanism of proinflammatory cytokine release and lung
injury and suggest that the Tn vaccine may be a promising
treatment modality against hyperoxia-induced lung injury in
neonates. Future studies are necessary to evaluate the direct
therapeutic effects of anti-Tn antibody on hyperoxia-induced
lung injury.
ETHICS STATEMENT
Animal care and experimental procedures were performed
in accordance with the guidelines of the Laboratory Animal
Care Committee of Taipei Medical University (LAC-2017-
0291). Sprague–Dawley rats (6 weeks old) were obtained
Frontiers in Immunology | www.frontiersin.org 8April 2019 | Volume 10 | Article 681

Chen et al. Immunization and Hyperoxia-Induced Lung Injury
from BioLASCO Taiwan Co., Ltd and were maintained
in a pathogen-free facility and air-conventional animal
housing on a 12-h light/dark cycle. The care and housing
of experimental animals were approved in accordance with the
guidelines of the Laboratory Animal Care Committee of Taipei
Medical University.
AUTHOR CONTRIBUTIONS
C-MC and JH: designed and performed the experiments; C-MC,
JH, and H-CC: analysis and interpretation of data and drafted
and approved the manuscript.
FUNDING
This study received grants from the Ministry of Science and
Technology in Taiwan (107-2314-B-038-060-MY2).
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fimmu.
2019.00681/full#supplementary-material
Supplement Figure 1 | Experimental design of the study timeline and rat
treatment groups.
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Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest.
Copyright © 2019 Chen, Hwang and Chou. This is an open-access article distributed
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