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Immunobiology
221
(2016)
377–386
Contents
lists
available
at
ScienceDirect
Immunobiology
jo
ur
nal
ho
me
page:
www.elsevier.com/locate/imbio
Expression
of
surfactant
proteins
SP-A
and
SP-D
in
murine
decidua
and
immunomodulatory
effects
on
decidual
macrophages
Shanmuga
Priyaa
Madhukarana,b,c,
Aghila
Rani
Koippallil
Gopalakrishnana,
Hrishikesh
Pandita,
Eswari
Dodagatta-
Marric,
Lubna
Kouserc,
Kaiser
Jamilb,
Fatimah
S.
Alhamland,
Uday
Kishorec,∗,
Taruna
Madana,∗
aDepartment
of
Innate
Immunity,
National
Institute
for
Research
in
Reproductive
Health,
Mumbai
400
012,
India
bCentre
for
Biotechnology
and
Bioinformatics,
School
of
Life
Sciences,
Jawaharlal
Nehru
Institute
for
Advanced
Studies,
Secunderabad,
Telangana,
India
cCentre
for
Infection,
Immunity
and
Disease
Mechanisms,
College
of
Health
and
Life
Science,
Brunel
University
London,
Uxbridge,
UB8
3PH,
United
Kingdom
dDepartment
of
Infection
and
Immunity,
King
Faisal
Specialist
Hospital
and
Research
Centre,
Riyadh,
Saudi
Arabia
a
r
t
i
c
l
e
i
n
f
o
Article
history:
Received
29
June
2015
Received
in
revised
form
12
September
2015
Accepted
14
September
2015
Available
online
16
September
2015
Keywords:
Murine
Decidua
Decidual
macrophages
SP-A
SP-D
Stromal
cells
Pregnancy
Parturition
a
b
s
t
r
a
c
t
Surfactant
proteins
SP-A
and
SP-D
are
pattern
recognition
innate
immune
molecules
that
belong
to
the
C-type
lectin
family.
In
lungs,
they
play
an
important
role
in
the
clearance
of
pathogens
and
control
of
inflammation.
SP-A
and
SP-D
are
also
expressed
in
the
female
reproductive
tract
where
they
play
an
important
role
in
pregnancy
and
parturition.
However,
the
role
of
SP-A
and
SP-D
expressed
at
the
feto-maternal
interface
(decidua)
remains
unclear.
Here,
we
have
examined
the
expression
of
SP-A
and
SP-D
in
the
murine
decidua
at
17.5
(pre-parturition)
and
19.5
dpc
(near
parturition)
and
their
effect
on
lipopolysaccharide
(LPS)-treated
decidual
macrophages.
SP-A
and
SP-D
were
localized
to
stromal
cells
in
the
murine
decidua
at
17.5
and
19.5
dpc
in
addition
to
cells
lining
the
maternal
spiral
artery.
Purified
pre-parturition
decidual
cells
were
challenged
with
LPS
with
and
without
SP-A
or
SP-D,
and
expression
of
F4/80
and
TNF-␣
were
measured
by
flow
cytometry.
On
their
own,
SP-A
or
SP-D
did
not
affect
the
percentage
of
F4/80
positive
cells
while
they
suppressed
the
percentage
of
TNF-␣
positive
cells.
However,
simultaneous
addition
of
SP-A
or
SP-D,
together
with
LPS,
reduced
TNF-␣
secreting
F4/80
positive
cells.
It
is
likely
that
exogenous
administration
of
SP-A
and
SP-D
in
decidua
can
potentially
control
infection
and
inflammation
mediators
during
spontaneous
term
labor
and
infection-induced
preterm
labor.
Thus,
the
presence
of
SP-A
and
SP-D
in
the
murine
decidua
is
likely
to
play
a
protective
role
against
intrauterine
infection
during
pregnancy.
©
2015
Elsevier
GmbH.
All
rights
reserved.
1.
Introduction
Intrauterine
infection
and
chorioamnionitis
are
very
common
complications
of
pregnancy
leading
to
stillbirth,
premature
birth,
and
neonatal
sepsis.
Chorioamnionitis
complicates
as
many
as
40–70%
of
preterm
births
due
to
premature
membrane
rupture
or
spontaneous
labor
and
up
to
13%
of
term
births
(Chang
et
al.,
2013).
Understanding
immunological
mechanisms
that
initiate
parturi-
tion
while
offering
a
defense
shield
at
the
feto-maternal
interface
can
help
devise
strategies
to
reduce
preterm
birth.
Surfactant
proteins,
SP-A
and
SP-D
are
collagenous
C-type
lectins
(also
called
collectins)
which
perform
a
range
of
innate
∗Corresponding
authors.
E-mail
addresses:
uday.kishore@brunel.ac.uk,
ukishore@hotmail.com
(U.
Kishore),
taruna
m@hotmail.com
(T.
Madan).
immune
functions
in
the
lungs,
including
clearance
of
pathogens
and
apoptotic/necrotic
cells,
regulation
of
inflammation,
and
prim-
ing
of
adaptive
immunity
(Kishore
et
al.,
2005,
2006;
Sano
and
Kuroki,
2005;
Nayak
et
al.,
2012).
SP-A
and
SP-D
are
26–36
KDa
and
43
KDa
proteins
in
size
that
assemble
further
to
form
high
molecular
weight
oligomeric
structure
of
630
KDa
and
520
KDa,
respectively
(Holmskov
et
al.,
2003).
SP-A
differs
from
SP-D
in
the
gene
organization,
structure,
ligand
binding
and
function
(Holmskov,
2000).
Their
primary
structure
comprises
of
an
N-
terminal
domain
with
cysteine
residues
for
interchain
disulphide
bond
formation,
a
C-terminal
carbohydrate
recognition
domain
(CRD),
the
alpha-helical
coiled
neck
with
amphipathic
helix,
a
collagen-like
domain
with
repeating
Gly-X-Y
and
hydroxyproline
residues
(Holmskov
and
Jensenius,
1993;
Haagsman
and
Diemel,
2001;
Kuroki
and
Sano,
1999).
SP-A
and
SP-D
bind
their
targets
mostly
via
CRDs
while
the
triple-helical
collagen
region
can
interact
with
CD91-
calreticulin
complex
on
the
cell
surface
of
phagocytic
http://dx.doi.org/10.1016/j.imbio.2015.09.019
0171-2985/©
2015
Elsevier
GmbH.
All
rights
reserved.
378
S.P.
Madhukaran
et
al.
/
Immunobiology
221
(2016)
377–386
cells,
leading
to
effector
mechanisms
such
as
phagocytosis,
super-
oxide
radical
generation,
and
cytokine
production
(Gardai
et
al.,
2003;
Wright,
2005).
In
lungs,
SP-A
and
SP-D
are
synthesized
and
secreted
by
alveo-
lar
type
II
and
Clara
cells
at
the
air-liquid
interface
of
the
surfactant
(Crouch
et
al.,
1992;
Voorhout
et
al.,
1992).
Expression
of
SP-A
and
SP-D
has
also
been
reported
in
extra
pulmonary
tissues
such
as
brain,
salivary
glands,
lachrymal
glands,
heart,
trachea,
kidney,
pan-
creas,
thymus,
spleen,
gall
bladder,
esophagus,
small
intestine,
large
intestine,
testis,
prostate
and
urinary
tract
(Madsen
et
al.,
2003;
Herías
et
al.,
2007;
Breuiller-Fouché
et
al.,
2010;
Nayak
et
al.,
2012;
Schicht
et
al.,
2015).
In
addition,
reproductive
tissues
have
also
been
shown
to
express
both
SP-A
and
SP-D
(Sati
et
al.,
2009;
Condon
et
al.,
2004;
Yadav
et
al.,
2011).
SP-A
and
SP-D
can
be
localized
within
the
fetal
membranes
(amniotic
epithelium
and
chorionic
membrane);
the
choriodecid-
ual
layer
of
the
late
pregnant
uterus;
cytotrophoblast,
intermediate
trophoblast
and
syncytiotrophoblast
of
early
gestation;
and
tro-
phoblast
of
late
normal
placental
villi
(Miyamura
et
al.,
1994;
Leth-Larsen
et
al.,
2004).
SP-D
level
in
the
amniotic
fluid
increases
gradually
from
0.11
g/ml
(14–16th
week
of
gestation)
to
26.3
g/ml
(38–42nd
week
of
gestation)
(Miyamura
et
al.,
1994;
Leth-Larsen
et
al.,
2004).
SP-A
shows
a
rise
from
3
g/ml
(30–31st
week
of
gestation)
to
24
g/ml
(40–41st
week
of
gestation)
near
the
term
(Miyamura
et
al.,
1994).
Expression
of
SP-A
in
pre-
and
post-menopausal
vaginal
stratified
squamous
epithelium
and
vagi-
nal
lavage
fluid
has
also
been
demonstrated
(MacNeill
et
al.,
2004).
Human
deciduaat
term
as
well
as
first
trimester
show
presence
of
SP-A
and
SP-D
(Snegovskikh
et
al.,
2011;
Madhukaran
et
al.
2015).
In
mouse,
SP-D
mRNA
and
protein
are
mainly
expressed
in
the
vagina,
uterus,
ovary,
cervix,
and
oviduct
(Akiyama
et
al.,
2002).
SP-D
expression
in
the
mouse
uterus
is
hormonally
regulated,
increasing
toward
estrus
and
decreasing
near
diestrus
(Oberley
et
al.,
2007;
Kay
et
al.,
2015).
Human
term
placental
tissues
express
both
SP-
A
and
SP-D
and
their
levels
alter
significantly
during
spontaneous
labor
(Yadav
et
al.,
2014).
Decidua
is
an
immunologically
privileged
site
that
bridges
the
maternal
and
fetal
immune
mechanisms
at
the
maternal-fetal
interface,
offering
protection
to
the
semi-allogenic
fetus
(Taglauer
et
al.,
2010;
Hsu
and
Nanan,
2014).
Within
decidua,
there
are
two
distinct
regions;
decidua
basalis
and
decidua
parietalis.
Decidua
basalis
is
embedded
into
the
placental
bed
invading
the
interstitial
trophoblast
while
decidua
parietalis
remains
in
contact
with
the
fetal
membrane
(Gomez-Lopez
et
al.,
2010).
Decidua
is
enriched
with
terminally
differentiated
macrophages
that
are
immuno-
suppressive
(Houser
et
al.,
2011).
Macrophages
are
the
second
most
predominant
leukocyte
population
(20–25%)
in
decidua
with
several
functions
from
early
until
late
gestation
(Trundley
and
Moffett,
2004;
Leonard
et
al.,
2006;
Gomez-Lopez
et
al.,
2010).
Their
number
increases
during
the
first
trimester
and
remains
constant
until
the
third
trimester;
however,
it
decreases
sig-
nificantly
prior
to
labor,
during
labor
and
postpartum
(Mackler
et
al.,
1999;
Shynlova
et
al.,
2013).
In
human,
early
pregnancy
decidua
has
∼50%,
while
term
pregnancy
decidua
has
∼20–30%
of
CD14+decidual
macrophages
(DMs)
(Gomez-Lopez
et
al.,
2014).
DMs
isolated
from
the
human
term
placenta
produce
a
consid-
erable
amount
of
TNF-␣
when
stimulated
with
LPS
(Singh
et
al.,
2005;
Gomez
et
al.,
1997).
Thus,
infiltration
of
DMs
in
the
mouse
decidua
has
been
considered
important
for
labor
cascade
(Hamilton
et
al.,
2012).
Infection
in
the
decidua
is
a
significant
threat
to
the
mother
and
the
fetus
during
pregnancy
(Mogensen,
2009).
Intra-
uterine
infection
and
consequent
inflammatory
response
have
been
associated
with
preterm
labor
(Burdet
et
al.,
2014).
Infec-
tion
during
pregnancy,
as
in
chorioamnionitis,
pyelonephritis,
and
chronic
deciduitis,
activates
DMs
via
LPS,
which
in
turn
gener-
ates
pro-inflammatory
TNF-␣
and
prostaglandin
F2␣in
the
decidua,
leading
to
preterm
labor
(Casey
et
al.,
1989;
Snegovskikh
et
al.,
2011).
Condon
et
al.
have
proposed
that
SP-A
secreted
from
fetal
lungs
can
activate
fetal
macrophages
(Condon
et
al.,
2004),
which
get
infiltrated
into
the
maternal
tissues
and
provoke
pro-inflammatory
response
via
increased
expression
of
IL-1
and
NF-B
that
initiates
labor
(Condon
et
al.,
2004).
This
highlights
the
importance
of
SP-A
in
the
cervical
ripening
and
uterine
contraction
leading
to
parturi-
tion.
SP-A
can
modulate
LPS-induced
signaling
via
TLRs
(Sano
et
al.,
1999;
Sano
et
al.,
2000;
Agrawal
et
al.,
2013).
Recently,
we
have
shown
that
SP-A
and
SP-D
are
expressed
by
stromal
cells
and
tro-
phoblasts
in
early
human
decidua
(Madhukaran
et
al.,
2015).Here,
we
show
the
expression
of
SP-A
and
SP-D
in
the
murine
decidua
pre
(17.5
dpc)
and
near
parturition
(19.5
dpc)
and
their
immunomod-
ulatory
effects
on
DMs
when
challenged
with
LPS.
2.
Materials
and
methods
2.1.
Ethics
statement
The
study
was
approved
by
the
institutional
animal
ethics
com-
mittee
(IAEC
no:
78/1999)
at
the
National
Institute
for
Research
in
Reproductive
Health,
Mumbai,
India.
All
procedures
were
carried
out
in
accordance
with
the
institutional
guidelines
for
the
care
and
use
of
experimental
animals.
2.2.
Animal
models
Inbred
strains
of
C57BL/6
female
and
male
mice
were
housed
in
a
humidity-controlled
animal
facility
under
standard
environmen-
tal
conditions
(12
h,
light/dark
cycle)
and
fed
ad
libitum.
Female
virgin
mice
(8–12
week
old)
were
housed
overnight
with
males
and
checked
for
the
presence
of
vaginal
plugs
the
next
morning
to
obtain
accurately
timed
pregnant
mice.
The
day
of
the
plug
forma-
tion
was
counted
as
0.5
post
coitus
(dpc).
Pregnant
mice
delivered
between
day
19
and
21.
Decidual
tissues
were
collected
on
ges-
tational
days
17.5
dpc
and
19.5
dpc.
Similarly,
mouse
fetal
lung
sample
was
collected
for
PCR.
2.3.
Isolation
of
murine
decidua
and
fetal
lungs
at
17.5
and
19.5
dpc
A
vertical
incision
was
made
in
the
abdomen
of
euthanized
(via
cervical
dislocation)
female
mice
under
sterile
conditions.
The
uter-
ine
horns
with
embryos
were
then
carefully
removed
and
washed
with
PBS
(Fig.
1A).
The
embryo
was
separated
from
the
uter-
ine
membrane
with
intact
placenta
(Fig
1B).
The
top
layer
of
the
embryo,
the
yolk
sac,
was
opened
along
the
anti-mesometrial
side.
Each
embryo
was
then
detached
from
its
placenta
and
rinsed
in
cold
PBS
(Fig
1C).
Decidua
parietalis,
was
identified
by
their
smooth,
grayish
solid
appearance
(Fig
1C).
Decidua
was
gently
removed
leaving
behind
the
placenta
and
decidua
spongiosa
which
is
dark
brown
in
color
with
spongy
appearance
(Fig
1D)
(Dudley
et
al.,
1993).
The
decidua
was
washed
several
times
with
ice-cold
PBS
to
minimize
blood
contamination
and
weighed
(Fig
1E).
Decid-
ual
tissues
at
17.5
dpc
and
19.5
dpc
were
cut
into
4–5
m
thick
sections,
fixed,
embedded
in
paraffin
wax,
stained
with
hema-
toxylin
and
eosin
for
histological
examination
(Fig
1F),
or
processed
for
immunohistochemistry.
In
all
cases,
decidualized
endometrial
stromal
cells
with
abundant
cytoplasm
and
vesicular
nuclei
were
observed.
Decidual
cells
were
round
to
polygonal
with
sharply
defined
cell
borders
and
single
nucleus
containing
small
but
promi-
nent
nucleolus
(The
observations
made
are
not
visible
from
the
Fig.
3,
it
requires
a
higher
magnification
to
show
nucleus
and
S.P.
Madhukaran
et
al.
/
Immunobiology
221
(2016)
377–386
379
Fig.
1.
Isolation
and
purification
of
decidual
tissue
and
cells.
(A)
Uterine
horn
separated
from
C57BL/6
pregnant
mice;
(B)
embryo
with
intact
placenta;
(C)
placenta
detached
from
the
embryo
and
embryo
sac;
(D)
decidual
tissue
macroscopically
separated
from
the
placenta;
(E)
isolated
decidual
tissues
in
PBS
(F)
Histological
examination
of
term
decidua
showing
decidualized
stromal
cells
and
abundant
cytoplasm
(pink)
and
nuclei
(blue)
stained
with
haematoxylin
and
eosin.
Black
arrow
represents
spongiotro-
phoblasts,
black
arrow
head
represents
giant
trophoblasts
cells,
yellow
arrow
head
represents
decidualized
stromal
cells
with
immune
cell
infiltration.
Scale
bar
=
100
m.
(For
interpretation
of
the
references
to
colour
in
this
figure
legend,
the
reader
is
referred
to
the
web
version
of
this
article.)
nucleolus).
Intact
fetal
lungs
were
harvested
on
ice.
All
tissues
were
rinsed
in
ice-cold
PBS.
2.4.
Immunohistochemical
staining
of
decidua
Immunohistochemistry
was
performed
using
5
m
paraffin
sec-
tions
over
Poly-Lysine
coated
slides.
The
decidual
sections
were
deparaffinized
and
rehydrated
followed
by
antigen
retrieval
with
0.1
M
sodium
citrate,
pH
6.0.
Blocking
was
carried
out
with
5%
(v/v)
normal
goat
serum
for
1
h
at
room
temperature.
Tissue
sections
were
probed
with
rabbit
anti-SP-A
and
SP-D
polyclonal
antibodies
(Abcam)
and
incubated
overnight
at
4◦C.
Following
three
washes
with
PBS,
sections
were
incubated
with
goat
anti-
rabbit
IgG
conjugated
to
horseradish
peroxidase
(Dako,
Denmark)
for
2
h
at
room
temperature.
The
sections
for
positive
antibody
binding
were
detected
with
chromogen
substrate
(AEC,
Dako),
then
380
S.P.
Madhukaran
et
al.
/
Immunobiology
221
(2016)
377–386
Fig.
2.
SDS-PAGE
(12%
w/v)
showing
(A)
rhSP-A
after
purification
on
maltose-agarose
column
following
denaturation
and
renaturation
procedure
of
inclusion
bodies,
Lane
2–4
showing
a
band
at
∼18
kDa
and,
(B)
rhSP-D
purified
after
folding
and
refolding
insoluble
protein
that
was
then
applied
to
the
maltose-agarose
column.
Peak
fractions
eluted
with
EDTA
(lane
2–4)
show
a
band
at
∼20
kDa.
counterstained
with
hematoxylin,
rehydrated
and
mounted.
Neg-
ative
control
sections
were
incubated
with
isotype-matched
IgG
at
the
same
concentration
as
the
primary
antibody.
2.5.
Real
time
RT-PCR
to
measure
SP-A
and
SP-D
mRNA
levels
in
decidua
Total
RNA
was
extracted
from
homogenized
decidual
tissues
at
17.5
and
19.5
dpc
in
TRIzol
reagent
(Genei,
Bangalore,
India)
using
chloroform
and
precipitated
with
ice-cold
isopropanol.
RNA
concentration
was
determined
spectrophotometrically
260
nm,
and
the
purity
was
estimated
using
A260/A280
ratio
via
a
nano-
spectrophotometer.
Subsequently,
2
g
of
total
RNA
was
reverse
transcribed
using
SuperScriptTM III
reverse
transcriptase
(Invitro-
gen).
The
resulting
cDNA
was
used
as
a
template
for
real-time
PCR
using
Bio-Rad
CFX96
TouchTM real-time
PCR
detection
sys-
tem
using
the
iQTM SYBR
Green
Supermix
(Bio-Rad,
Hercules,
CA,
USA).
Primer
sequences
pertaining
to
mouse
SP-A,
SP-D
and
18s
rRNA
(housekeeping
gene
control)
are
shown
in
Table
1.
Relative
gene
expression
between
SP-A
and
SP-D
in
decidua
and
reference
mouse
fetal
lung
sample
at
17.5
dpc
and
19.5
dpc
was
determined
by
comparative
2−Ct method.
2.6.
Expression
and
purification
of
rhSP-A
and
rhSP-D
Recombinant
forms
of
human
SP-A
and
SP-D
containing
homotrimeric
neck
and
lectin
domains
were
expressed
in
Escherichia
coli
BL21
(DE3)
pLysS
(Life
Technologies,
UK)
(Karbani
et
al.,
2014;
Dodagatta-Marri
et
al.,
2014;
Singh
et
al.,
2003).
The
bacterial
cells
were
grown
in
Luria-Bertani
medium
(500
ml)
con-
taining
(100
g/ml)
and
chloramphenicol
(50
g/ml)
over
a
37 ◦C
incubator
shaker,
until
an
A600
of
0.6
was
reached.
Cells
were
induced
with
0.4
mM
isopropyl
-D-thiogalactoside
(IPTG)
(Sigma,
Poole,
UK)
for
3
h
at
37 ◦C
and
then
pelleted
via
centrifugation
(4500
rpm,
4◦C,
10
min).
The
cell
pellet
was
lysed
via
stirring
in
the
lysis
buffer
(50
mM
Tris–HCL
pH
7.5,
200
mM
NaCl,
5
mM
EDTA,
0.1%
v/v
Triton
X-100,
0.1
mM
PMSF,
50
g
lysozyme)
for
1
h,
and
then
sonicated
over
ice
using
a
Soniprep
150
(MSE,
London,
UK)
at
60
Hz
for
30
s
with
an
interval
of
2
min
(12
cycles).
The
sonicate
was
centrifuged
at
12000
rpm
for
15
min
and
the
inclusion
bod-
ies
were
solubilized
in
50
ml
buffer
A
(50
mM
Tris–HCl
pH
7.5,
and
100
mM
NaCl)
with
10
mM
2-mercaptoethanol
(Bio-Rad,
Hertford-
shire,
UK)
and
8
M
urea
for
1
h
at
4◦C.
The
soluble
fraction
was
dialysed
against
a
gradient
of
buffer
A
containing
4
M
urea,
2
M
urea,
and
1
M
urea
for
2
h
at
each
urea
concentration.
The
dialysate
was
finally
dialyzed
against
affinity
buffer
(50
mM
Tris–HCl
pH
7.5,
100
mM
NaCl,
10
mM
CaCl2)
with
2
changes
and
then
cen-
trifuged
(10,000
rpm,
10
min,
4◦C).
The
supernatant
was
loaded
onto
a
maltose–agarose
column
(Sigma;
5
ml)
and
the
column
was
washed
with
3
column
volumes
of
affinity
buffer;
the
bound
protein
was
eluted
with
buffer
A
containing
10
mM
EDTA.
Peak
fractions
were
analyzed
for
purity
on
SDS-PAGE
(Fig.
2).
For
LPS
removal,
5
ml
of
Polymyxin
B
agarose
gel
(Sigma,
Poole,
UK)
was
packed
in
a
20
ml
Bio-Rad
column
and
washed
with
50
ml
of
1%
sodium
deoxy-
cholate.
The
matrix
was
then
further
rinsed
with
50
ml
of
sterile
distilled
water
to
completely
remove
sodium
deoxycholate.
Affin-
ity
purified
rhSP-D
or
rhSP-A
fractions
were
applied
to
Polymyxin
B
columns
for
at
4◦C.
The
recombinant
proteins
were
collected
in
the
flow
through
as
1
ml
fractions.
The
endotoxin
levels
were
determined
by
QCL-1000
Limulus
amebocyte
lysate
kit
(BioWhit-
taker,
Walkersville,
MD,
USA),
which
was
found
to
be
∼5
pg
g−1
of
rhSP-D
and
∼4
pg
g−1of
rhSP-A.
Table
1
Primers
used
for
amplification
of
cDNAs.
Gene
Target
Annealing
Temperature
Product
Size
(Base
pairs)
5#8∼8#
Sequences
(Forward
Primer)
5#8∼8#
Sequences
(Reverse
Primer)
SP-A
60 ◦C
225
ACCTGGATGAGGAGCTTCAGACTGC
TGCTTGCGATGGCCTCGTTCT
SP-D
66 ◦C
156
CAAAAGGCTCCACAGGCCCCA
CAGCACTGTCTGGAAGCCCGC
18s
64 ◦C
175
GGAGAGGGAGCCTGAGAAAC
CCTCCAATGGATCCTCGTTA
S.P.
Madhukaran
et
al.
/
Immunobiology
221
(2016)
377–386
381
Fig.
3.
Immunohistochemical
localization
of
SP-A
and
SP-D
in
the
murine
decidua
at
17.5
dpc
(A
and
D)
and
19.5
dpc
(B
and
E).
SP-A
and
SP-D
(brown)
were
observed
in
the
decidua
stroma
as
well
as
in
and
around
the
spiral
artery
and
blood
vessels.
Intense
SP-D
(D)
and
(E)
staining
was
detected
at
17.5
and
19.5
dpc
when
compared
to
SP-A.
In
the
negative
control
where
the
primary
antibody
was
excluded
(C
and
F),
no
staining
was
observed.
(n
=
6,
Original
magnification
20×,
Scale
Bar:
20
m.
(For
interpretation
of
the
references
to
colour
in
this
figure
legend,
the
reader
is
referred
to
the
web
version
of
this
article.)
2.7.
Isolation
of
decidual
cells
Decidual
cells
were
isolated
and
purified
as
previously
described
(Vince
et
al.,
1990;
Singh
et
al.,
2005).
Briefly,
decidua
was
finely
minced
in
PBS
with
constant
stirring
to
remove
most
of
the
remaining
blood
and
digested
using
collagenase
type
IV
(Gibco)
in
RPMI-1640
medium.
The
cell
suspension
was
passed
through
40
m
filters
to
dissociate
remaining
cell
clusters
and
then
cen-
trifuged
at
1500
rpm
for
7
min.
The
cell
pellet
was
first
washed
with
PBS
and
then
with
RPMI-1640
containing
10%
v/v
heat-inactivated
FCS.
The
cell
suspension
was
examined
under
the
light
microscope
and
counted
using
hemocytometer.
2.8.
LPS
treatment
of
decidual
cells
comprising
of
DMs
pre-treated
with
and
without
rhSP-A
or
rhSP-D
In
order
to
address
the
question
whether
rhSP-A
or
rhSP-D
can
suppress
the
pro-inflammatory
effect
of
LPS,
decidual
cells
(1
×
106/ml)
in
RPMI-1640
containing
10%
FCS
were
pretreated
for
1
h
at
37 ◦C
with
5
or
10
g
of
rhSP-A,
rhSP-D
or
LPS
(100
ng)
(derived
from
E.
coli
strain
011:B4).
Cells
were
then
carefully
washed
with
PBS
and
then
challenged
with
LPS
or
PBS
(Control),
respectively
for
an
additional
6
h
at
37 ◦C.
This
was
followed
by
dual
staining
of
decidual
cells
with
F4/80-RPE
(F4/80
is
a
cell
surface
marker
for
murine
macrophages)
and
measurement
of
intracellular
TNF-␣
in
permeabilised
decidual
cells.
Prior
to
intracellular
staining,
cells
were
washed
with
ice-cold
PBS
and
incubated
for
cell
surface
staining
using
anti-mouse
F4/80-
RPE
(phycoerythrin)
(30
min,
4◦C).
Cells
were
then
fixed
with
4%
Para
formaldehyde
and
incubated
at
4◦C
for
15
min
in
dark.
Cells
were
washed
again
and
resuspended
in
0.1%
saponin
and
incubated
for
20
min
over
ice
in
dark
to
permeabilize
the
cells.
Cells
were
then
washed
and
incubated
with
anti-mouse
TNF-␣
FITC
(Abcam)
for
30
min
at
4◦C
to
IgG
isotype-PE
and
FITC
and
untreated
cells
were
used
to
adjust
appropriate
scatter
for
FACs
analysis.
Finally,
cell
suspensions
were
washed,
resuspended
in
PBS
and
analyzed
using
Flow
cytometer
Aria
III
(Beckton
Dickinson).
The
decidual
cell
population
was
gated
according
to
the
scatter
plot
as
deter-
mined
by
the
characteristic
forward
scatter
(FS)
and
side
scatter
(SC).
The
analysis
involved
decidual
cells
which
were
F4/80+,
TNF-
␣+,
and
TNF-␣+/F4/80+.
The
murine
DMs
(F4/80)
expressing
TNF-␣
were
evaluated
in
terms
of
the
percentage
of
TNF-␣
expression
by
382
S.P.
Madhukaran
et
al.
/
Immunobiology
221
(2016)
377–386
decidual
cells
and
F4/80
positive
DMs,
or
by
mean
fluorescence
intensity
(MFI).
Decidual
cells,
before
and
after
stimulation
with
LPS,
were
washed
and
stained
with
Trypan
blue
for
10
min
and
then
examined
by
light
microscopy
to
assess
the
cell
viability.
The
cell
viability
was
∼95%.
Out
of
1,
2,
4,
and
6
h
incubation
with
LPS,
surfactant
proteins
showed
most
significant
suppression
of
the
6
h-
LPS
induced
TNF-
␣
in
decidual
cells.
2.9.
Experimental
replicates
and
statistical
analysis
Data
analysis
was
performed
using
Statistical
Package
for
Social
Sciences
(SPSS,
Version
17).
Student’s
t-test
was
employed
to
compare
the
mRNA
expression
levels
of
SP-A
and
SP-D
between
17.5
dpc
and
19.5
dpc
decidual
tissues.
Analysis
of
Variance
(ANOVA)
was
performed
using
StatPlus
(Version
5.3.5.1)
to
deter-
mine
whether
the
means
of
outcomes
were
different
by
treatment
and
assess
the
statistical
significance
for
the
FACS
analysis.
All
val-
ues
are
expressed
as
mean
values
of
experimental
replicates
±
SEM
obtained
from
triplicates,
and
the
data
are
representatives
of
three
independent
experiments.
P
<
0.05
was
considered
to
be
statisti-
cally
significant.
3.
Results
3.1.
SP-A
and
SP-D
proteins
in
the
murine
decidua
at
term
We
investigated
SP-A
and
SP-D
expression
at
the
protein
level
using
immunohistochemistry.
In
decidual
tissues,
we
observed
dif-
fused
SP-A
and
SP-D
proteins
at
both
17.5
dpc
and
19.5
dpc
which
were
detected
in
a
range
of
decidual
stromal
cells
as
well
as
mater-
nal
spiral
artery
in
close
proximity
to
trophoblasts
(Fig.
3).
SP-A
and
SP-D
were
also
localized
in
the
extracellular
space
within
the
decidua
at
17.5
dpc
and
19.5
dpc
(Fig.
3).
This
was
confirmed
by
using
negative
control
that
were
incubated
with
secondary
anti-
body
alone
(Fig
3C
and
F).
3.2.
Expression
of
SP-D,
but
not
SP-A,
by
term
decidual
cells
To
ascertain
the
source
of
SP-A
and
SP-D
in
the
term
decidua,
primers
were
designed,
and
expression
was
assessed
by
real-time
RT-PCR
(Fig.
4).
Murine
fetal
lungs
that
were
used
as
positive
con-
trol,
as
expected,
expressed
both
SP-A
as
well
as
SP-D.
A
significant
expression
of
SP-D
mRNA
was
detected
in
the
decidua
at
17.5
and
19.5
dpc.
In
contrast,
mRNA
expression
levels
for
SP-A
gene
were
low
and
undetectable
in
the
decidua
at
17.5
and
19.5
dpc,
but
its
expression
was
high
in
fetal
lungs
(Figs.
4
and
5).
Remark-
ably,
decidua
at
17.5
dpc
showed
significantly
higher
expression
of
SP-D
when
compared
to
19.5
dpc
decidua
(p
<
0.05).
To
confirm
the
specificity
of
SP-A
and
SP-D
amplification,
the
amplified
PCR
prod-
ucts
were
visualized
on
a
2%
agarose
gel
and
stained
with
ethidium
bromide.
The
results
revealed
specific,
amplified
SP-D
mRNA
17.5 dpc19.5 dpc
0
1
2
3
4
5
6
7
Fold change w.r.t
random calibrator
SP-A
SP-D
**
Fig.
4.
Expression
levels
of
SP-A
and
SP-D
mRNA
in
the
murine
decidua
at
17.5
and
19.5
dpc.
Relative
expression
levels
of
SP-A
and
SP-D
transcripts
normalized
to
the
transcript
levels
of
18s
rRNA
present
in
each
sample
were
determined
by
real-
time
PCR.
The
results
represent
the
mean
of
three
individual
experiments;
error
bars
represent
S.D.
The
gene
expression
is
presented
as
fold-change
calculated
by
comparative
Ct
analysis.
**
p
<
0.05.
Fig.
5.
Gene
expression
levels
of
SP-A
(225
bp),
SP-D
(156
bp)
in
the
term
decidua.
2
g
total
RNA
in
decidua
were
used
for
RT-PCR
and
amplified
products
were
sep-
arated
by
agarose
gel
electrophoresis.
Lung
sample
was
used
as
positive
control
for
SP-A
and
SP-D
while
18s
(175
bp)
mRNA
level
was
used
as
an
internal
control.
Lane
1:
Ladder
(100
bp);
Lane
2:
18s;
Lane
3:
18s
NTC;
Lane
4:
SP-A
positive
control
(lung);
Lane
5:
SP-A
decidua;
Lane
6:
SP-A
NTC;
Lane
7:
SP-D
positive
control
(lung);
Lane
8:
SP-D
decidua;
Lane
9:
SP-D
NTC.
(n
=
3).
(NTC
=
No
template
control).
product
(156
bp)
in
term
decidua
and
controls
(Fig
5).
There
is
a
clear
difference
in
the
bands
observed
in
lane
4
and
5
in
Fig.
5.
Lane
4
shows
expression
of
SP-A
in
the
fetal
lung
whereas,
decidua
in
lane
5
reveals
no
band
of
225
bp
corresponding
to
SP-A
mRNA.
3.3.
Effect
of
SP-A
and
SP-D
on
LPS
challenged
murine
decidual
cells
To
investigate
the
effects
of
SP-A
and
SP-D
on
DM
(the
largest
population
of
immune
cells
in
mouse
decidua),
we
measured
the
TNF-␣
produced
by
murine
F4/80
macrophages
at
17.5
dpc
decidua
when
challenged
with
rhSP-A
or
rhSP-D
or
LPS.
We
observed
4.0
±
0.6
%
F4/80
positive
macrophages
and
11.6
±
1.3
%
TNF-␣
pro-
ducing
decidual
cells
at
17.5
dpc
(Fig
6A,
Table
2).
Interestingly,
expression
of
intracellular
TNF-␣
production
in
DMs
was
signif-
icantly
reduced
by
both
rhSP-A
and
rhSP-D
in
a
dose-dependent
manner
(Fig
6A,
B,
and
Table
2).
LPS
challenge
induced
an
inflam-
matory
response
within
the
decidual
cells
by
elevating
the
levels
of
Table
2
In
vitro
effect
of
rhSP-A,
rhSP-D
on
LPS
induced
TNF-␣
production
by
17.5
dpc
decidual
macrophages.
Decidual
cells
%
of
F4/80
cells
%
of
total
TNF-␣
production
%
of
TNF-␣
by
Macrophages
Control
or
untreated
cells
4.0
±
0.6
11.6
±
1.3
4.4
±
0.7
LPS
(100
ng)
9.9
±
2.4
29.1
±
3.4
8.7
±
1.3
SP-A
(5
g)
4.2
±
1.1
9.1
±
1.8
3.6
±
1.5
SP-A
(10
g)
3.2
±
2.1
7.9
±
2.1
1.8
±
0.4
SP-A
(5
g)
+
LPS
(100
ng)
7.4
±
2.3
23.2
±
3.2
6.2
±
2.3
SP-A
(10
g)
+
LPS
(100
ng)
9.7
±
1.7
14.2
±
2.4
5.8
±
0.8
SP-D
(5
g)
3.9
±
2.1
7.6
±
1.5
2.3
±
1.2
SP-D
(10
g)
3.7
±
0.6
11.2
±
2.1
1.7
±
0.3
SP-D
(5
g)
+
LPS
(100
ng)
3.9
±
0.6
16.7
±
1.7
2.9
±
1.2
SP-D
(10
g)
+LPS
(100
ng)
3.1
±
1.3
12.2
±
1.2
1.9
±
0.7
S.P.
Madhukaran
et
al.
/
Immunobiology
221
(2016)
377–386
383
Fig.
6.
Intracellular
TNF-␣
production
by
murine
decidual
macrophages
(pre-parturition,
17.5
dpc).
Fresh
murine
decidual
cells
(1
×
106/ml)
were
pretreated
with
5
or
10
g
of
rhSP-A,
rhSP-D
or
LPS
(100
ng).
Cells
were
then
carefully
washed
with
PBS
and
then
challenged
with
LPS
or
PBS
(control)
for
an
additional
6
h
at
37 ◦C
(A)
Flow
cytometric
analysis
of
F4/80+TNF-
␣+decidual
cells.
(a)
Untreated;
(b)
LPS
(100
ng);
(c)
rhSP-A
(5
g);
(d)
rhSP-A
(10
g);
(e)
rhSP-A
(5
g)
+
LPS
(100
ng);
(f)
rhSP-A
(10
g)
+
LPS
(100ng);
(g)
rhSP-D
(5
g);
(h)
rhSP-D
(10
g);
(i)
rhSP-D
(5
g)
+
LPS
(100
ng),
and
(j)
rhSP-D
(10
g)
+
LPS
(100
ng)
(B)
The
mean
percentage
of
total
TNF-␣+F4/80+and
TNF-␣+decidual
cells
in
various
experimental
conditions.*
p
<
0.05
with
respect
to
the
control
and
#
p
<
0.05
with
respect
to
the
LPS
treated
decidual
cells.
384
S.P.
Madhukaran
et
al.
/
Immunobiology
221
(2016)
377–386
TNF-␣
(Fig
6A,
B).
F4/80
positive
cells
were
higher
(9.9
±
2.4)
after
LPS
challenge,
when
compared
to
untreated
controls
(4.0
±
0.6),
indicating
an
increase
in
F4/80
positive
cells,
a
plausible
major
source
for
LPS
induced
TNF-␣
production
within
the
decidua.
The
percentage
of
F4/80+TNF-␣+cells
reduced
in
decidua
in
a
dose
responsive
manner
from
8.7%
(in
response
to
LPS
stimula-
tion
(100
ng)
to
1.8
%
with
10
g
rhSP-A,
3.6%
with
5
g
rhSP-A,
1.7%
with
10
g
rhSP-D
and
2.3%
with
5
g
rhSP-D.
Interestingly,
the
proportion
of
total
TNF-␣
producing
decidual
cells
were
also
significantly
reduced,
from
29.1%
upon
LPS
stimulation
to
7.9%
in
LPS
challenged
decidual
cells
treated
with
SP-A
(10
g),
9.1%
in
decidual
cells
treated
with
SP-A
(5
g),
7.6%
in
decidual
cells
treated
with
SP-D
(10
g)
and
11.2%
in
decidual
cells
treated
with
SP-D
(5
g)
(Fig
6B).
Thus
exogenous
rhSP-A
and
rhSP-D
significantly
reduced
LPS
induced
TNF-␣
release
in
decidual
cells
and
in
the
F4/80
positive
decidual
macrophage
cells
(Fig
6A
and
B).
4.
Discussion
SP-A
and
SP-D
are
pattern
recognition
innate
immune
molecules
that
modulate
inflammation
and
control
infection
in
lungs
(Kishore
et
al.,
2006;
Nayak
et
al.,
2012).
They
can
opsonize
and
clear
the
pathogens
via
phagocytosis
(Pastva
et
al.,
2007;
Kerrigan
and
Brown,
2009).
During
pathogen
elimination,
the
CRD
region
of
SP-
A
and
SP-D
is
involved
in
opsonization
while
the
collagenous
tail
interacts
with
the
calreticulin/CD91
receptor
on
immune
cells
to
mediate
pro-inflammatory
response
when
not
interacting
with
the
ligands,
the
CRDs
bind
to
signal
inhibitory
regulatory
pro-
tein
alpha
(SIRP-␣)
to
create
a
non-inflammatory
response
thereby
contributing
to
immune
homeostasis
(Gardai
et
al.,
2003).
This
dual
role
of
SP-A
and
SP-D
may
be
particularly
critical
in
the
onset
of
parturition
as
well
as
preterm
labor
associated
with
intrauter-
ine
infection.
Due
to
obvious
limitations
of
using
human
decidual
tissues
at
pre-parturition
and
near
parturition
stages,
we
used
C57BL/6
mice
to
examine
the
role
of
SP-A
and
SP-D
in
the
decidua
at
17.5
dpc
and
19.5
dpc.
Although
the
reproductive
events
are
species-specific,
the
functions
of
the
mouse
and
human
placenta
are
remarkably
similar,
thus
mouse
model
serves
as
an
essen-
tial
research
tool
for
studying
complicated
pregnancy
(Georgiades
et
al.,
2002).
The
data
presented
here
shows
that
(1)
the
levels
of
SP-
D
in
the
murine
decidua
decreases
near
parturition;
(2)
low
or
no
SP-A
transcript
but
presence
of
SP-A
protein
in
the
murine
decidua
during
pre
and
near
parturition;
(3)
and
the
presence
of
SP-A
and
SP-D
significantly
decreases
pro-inflammatory
cytokine
TNF-
␣
production
by
LPS
challenged
decidual
macrophages.
This
study
suggests
that
SP-A
and
SP-D
may
have
a
crucial
role
in
controlling
infection-induced
inflammatory
response
during
parturition.
Our
study
showed
no
SP-A
mRNA
expression
in
murine
decidua
at
17.5
dpc
and
19.5
dpc.
However,
SP-A
protein
expression
was
readily
detected
in
decidua
by
immunohistochemistry.
Our
results
raise
the
possibility
that
the
extracellular
SP-A
protein
expres-
sion
observed
by
IHC
in
the
decidua
is
likely
to
be
originating
from
the
fetal
lungs
and
amniotic
fluid
at
17.5
dpc
and
19.5
dpc.
A
previous
murine
study
by
Condon
et
al.,
have
documented
the
pro-inflammatory
effect
of
fetal
lung
SP-A.
The
amniotic
fluid
(AF)
SP-A
from
the
fetal
lung
is
proposed
to
act
as
a
hormone
that
sig-
nals
parturition
by
activating
AF
macrophages,
which
migrate
into
the
uterus
and
trigger
inflammatory
response,
leading
to
influx
of
leucocytes
for
uterine
contraction
and
onset
of
labor
(Condon
et
al.,
2004).
Studies
on
human
decidua,
however,
demonstrated
via
immunohistochemistry
that
SP-A
level
increases
in
decidual
stro-
mal
cells
before
labor
and
then
decreases
after
labor
in
the
decidua
(Snegovskikh
et
al.,
2011;
Yadav
et
al.,
2014).
The
decreased
SP-A
expression
has
been
suggested
to
stimulate
the
anti-inflammatory
cytokine
in
the
amnion
(Lee
et
al.,
2010).
Levels
of
SP-D
transcripts
in
murine
decidua
were
decreasing
toward
the
end
of
the
gestation
during
the
onset
of
parturition.
However,
these
find-
ings
are
not
similar
to
our
recent
study
with
human
term
decidua
that
demonstrated
an
increased
SP-D
protein
expression
during
spontaneous
labor
(Yadav
et
al.,
2014).
SP-D
restricts
Chlamydia
tra-
chomatis
infection
in
cervical
epithelial
cells
(Oberley
et
al.,
2004).
Thus,
we
speculate
that
an
increased
synthesis
of
decidual
SP-D
at
17.5
dpc
may
be
responsible
for
pregnancy
maintenance
underly-
ing
its
anti-inflammatory
property
while
the
gestational
decrease
of
decidual
SP-D
at
19.5
dpc
acts
as
a
hormonal
stimulus
for
initiating
the
inflammatory
events
crucial
for
cervical
remodeling
and
myometrial
contraction
leading
to
parturition.
Systemic
LPS
challenge
on
16th
and
17th
day
of
gestation
increases
the
expression
of
TLRs,
SP-A,
SP-D
and
cytokines
in
the
uterus.
These
uterine-derived
pro-inflammatory
mediators
can
induce
preterm
birth
in
mice
(Salminen
et
al.,
2008).
There
is
evidence
to
suggest
that
intrauterine
injection
of
SP-A
can
down-regulate
LPS
induced
inflammatory
ligand
TLRs,
(Agrawal
et
al.,
2013).
LPS
challenge
in
SP-A
and
SP-D
over-expressing
mice
increases
the
expression
of
SP-A,
SP-D,
and
TNF-␣
and
decreases
IL-10
expression
in
the
gestational
tissues
of
the
fetus
(Salminen
et
al.,
2011,
2012).
The
increased
fetal
SP-A
and
SP-D
levels
can
potentially
modulate
LPS-induced
inflammatory
response.
Thus,
it
was
of
interest
to
examine
the
in
vitro
modulatory
effect
of
SP-A
and
SP-D
on
LPS
challenged
decidual
cells
and
DMs
near
parturi-
tion.
An
increased
inflammatory
response
following
intrauterine
infection
can
induce
labor
leading
to
preterm
birth.
Therefore,
we
considered
that
SP-A
and
SP-D
expressed
in
decidua
may
play
a
role
in
preventing
infection-induced
preterm
labor.
Our
in
vitro
observations
suggest
that
SP-A
and
SP-D
are
capable
of
regulat-
ing
the
LPS-induced
TNF-␣
production
by
decidual
cells,
which
may
be
critical
for
containing
intra-uterine
infection
while
negating
the
possible
effects
of
TNF-␣
and
other
pro-inflammatory
media-
tors.
These
effects
of
SP-A
and
SP-D
could
be
plausibly
mediated
by
two
mechanisms.
SP-A
and
SP-D
are
known
to
directly
interact
with
LPS
and
inhibit
interaction
of
LPS
with
its
cellular
receptors,
thereby
reducing
the
pro-inflammatory
signaling
(Sano
et
al.,
1999,
2000;
Yamazoe
et
al.,
2008).
Further,
during
the
pre-treatment
SP-
A
and
SP-D
may
interact
with
inhibitory
receptors
on
decidual
cells
leading
to
their
reduced
response
to
LPS
challenge
(Gardai
et
al.,
2003).
Thus,
the
presence
of
SP-A
and
SP-D
in
decidua
may
be
critical
for
pregnancy
maintenance
and
protection
of
fetus
against
infection.
It
is
possible
that
abnormal
levels
of
SP-A
and
SP-D
in
decidua
near
parturition
may
contribute
to
fetal
mortality
in
infection-induced
preterm
labor.
Acknowledgements
We
would
like
to
thank
the
facilities
provided
by
National
Institute
for
Research
in
Reproductive
Health,
Mumbai
and
Indian
Council
of
Medical
Research
for
their
support.
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