Intestinal immunity in hypopituitary dwarf mice: effects of age


Wang, X.; Darcy, J.; Cai, C.; Jin, J.; Bartke, A.; Cao, D.

Aging 10(3): 358-370

2018


Hypopituitary dwarf mice demonstrate advantages of longevity, but little is known of their colon development and intestinal immunity. Herein we found that Ames dwarf mice have shorter colon and colonic crypts, but larger ratio of mesenteric lymph nodes (MLNs) over body weight than age-matched wild type (WT) mice. In the colonic lamina propria (cLP) of juvenile Ames mice, more inflammatory neutrophils (Ā: 0.15% vs. 0.03% in WT mice) and monocytes (Ā: 7.97% vs. 5.15%) infiltrated, and antigen presenting cells CD11c+ dendritic cells (Ā: 1.39% vs. 0.87%), CD11b+ macrophages (Ā: 3.22% vs. 0.81%) and gamma delta T (γδ T) cells (Ā: 5.56% vs. 1.35%) were increased. In adult Ames dwarf mice, adaptive immune cells, such as IL-17 producing CD4+ T helper (Th17) cells (Ā: 8.3% vs. 4.7%) were augmented. In the MLNs of Ames dwarf mice, the antigen presenting and adaptive immune cells also altered when compared to WT mice, such as a decrease of T-regulatory (Treg) cells in juvenile Ames mice (Ā: 7.7% vs.10.5%), but an increase of Th17 cells (Ā: 0.627% vs.0.093%). Taken together, these data suggest that somatotropic signaling deficiency influences colon development and intestinal immunity.

Research
Paper
Intestinal
immunity
in
hypopituitary
dwarf
mice:
effects
of
age
Xin
Wang
1
,
Justin
Darcy
1
'
2
,
Chuan
Cai
3
,
Junfei
Jin
4
,
Andrzej
Bartke
2
,
Deliang
Cao
1
'
3
1
Department
of
Medical
Microbiology,
Immunology,
and
Cell
Biology,
Southern
Illinois
University,
School
of
Medicine,
Springfield,
IL
62702,
USA
2
Department
of
Internal
Medicine,
Southern
Illinois
University,
School
of
Medicine,
Springfield,
IL
62702,
USA
3
Division
of
Stem
Cell
Regulation
and
Application,
State
Key
Laboratory
of
Chinese
Medicine
Powder
and
Medicine
Innovation
in
Hunan
(incubation),
Hunan
University
of
Chinese
Medicine,
Changsha,
Hunan
410208,
China
4
China-USA
Lipids
in
Health
and
Disease
Research
Center,
Guilin
Medical
University,
Guilin,
541001,
Guangxi,
China
Correspondence
to:
Deliang
Cao;
email:
dcao@siumed.edu
Keywords:
aging,
dwarfism,
colonic
development,
intestinal
immunity,
immune
cells
Received:
January
2,
2018
Accepted:
February
23,
201
Published:
March
2,
2018
Copyright:
Wang
et
al.
This
is
an
open-access
article
distributed
under
the
terms
of
the
Creative
Commons
Attribution
License
(CC
BY
3.0),
which
permits
unrestricted
use,
distribution,
and
reproduction
in
any
medium,
provided
the
original
author
and
source
are
credited.
ABSTRACT
Hypopituitary
dwarf
mice
demonstrate
advantages
of
longevity,
but
little
is
known
of
their
colon
development
and
intestinal
immunity.
Herein
we
found
that
Ames
dwarf
mice
have
shorter
colon
and
colonic
crypts,
but
larger
ratio
of
mesenteric
lymph
nodes
(MLNs)
over
body
weight
than
age-matched
wild
type
(WT)
mice.
In
the
colonic
lamina
propria
(cLP)
of
juvenile
Ames
mice,
more
inflammatory
neutrophils
(A:
0.15%
vs.
0.03%
in
WT
mice)
and
monocytes
(A:
7.97%
vs.
5.15%)
infiltrated,
and
antigen
presenting
cells
CD11c+
dendritic
cells
(A:
1.39%
vs.
0.87%),
CD11b+
macrophages
(A:
3.22%
vs.
0.81%)
and
gamma
delta
T
(y6
T)
cells
(A:
5.56%
vs.
1.35%)
were
increased.
In
adult
Ames
dwarf
mice,
adaptive
immune
cells,
such
as
IL-17
producing
CD4+
T
helper
(Th17)
cells
(A:
8.3%
vs.
4.7%)
were
augmented.
In
the
MLNs
of
Ames
dwarf
mice,
the
antigen
presenting
and
adaptive
immune
cells
also
altered
when
compared
to
WT
mice,
such
as
a
decrease
of
T-regulatory
(Treg)
cells
in
juvenile
Ames
mice
(A:
7.7%
vs.10.5%),
but
an
increase
of
Th17
cells
(A:
0.627%
vs.0.093%).
Taken
together,
these
data
suggest
that
somatotropic
signaling
deficiency
influences
colon
development
and
intestinal
immunity.
INTRODUCTION
Ames
dwarf
mice
possess
a
spontaneous
Prophet
of
Pituitary
Factor
1
(Propl)
loss-of-function
mutation.
The
mutation
of
the
Prop
1
gene
results
in
the
lack
of
differentiation
of
endocrine
cell
lineages
(somatotrophs,
lactotrophs
and
thyrotrophs)
in
the
anterior
pituitary.
Therefore,
Ames
dwarf
mice
are
deficient
in
growth
hormone
(GH)
and
insulin-like
growth
factor
1
(IGF-1),
thyroid-stimulating
hormone
(TSH),
the
thyroid
hormones
(THs),
and
prolactin
(PRL)
[1].
Particularly
important
is
the
deficiency
of
GH and
IGF-1
signaling
(collectively
referred
to
as
somatotropic
signaling)
[2],
which
is
believed
to
be
the
principle
force
behind
the
approximately
50%
increase
in
longevity
observed
in
Ames
dwarf
mice
(depending
on
sex
and
diet)
[3].
Mechanisms
that
are
responsible
for
the
longevity
of
Ames
dwarf
mice
may
include
improved
antioxidant
defense,
enhanced
insulin
sensitivity
and
reduced
insulin
levels,
reduced
inflammation
and
cell
senescence,
and
greater
stress
resistance
[4].
However,
it
is
unclear
how
the
somatotropic
signaling
(GH/IGF-1)
defect
affects
the
colon
development
and
intestinal
immunity
in
these
mice.
Beyond
the
main
role
of
promoting
linear
growth
and
metabolism,
GH
has
important
effects
on
the
immune
system.
For
instance,
GH
can
interact
with
B
and
T
lymphocytes
[5],
promote
thymic
growth
and
T
cell
development,
improve
T
cell
function,
and
enhance
the
www.aging-us.com
358
AGING
immune
response
[6,
7],
playing
an
important
role
in
homeostasis
of
immune
system.
High
endogenous
GH
levels
inhibit
specific
antibody
(Ab)
production
and
peripheral
T
cell
populations,
but
do
not
impact
peripheral
B
cell
number,
Th2
cell
population,
and
the
production
of
interleukin-4
(IL-4)
and
IFN-y
[8].
GH
and
IGF-I
are
involved
in
regulation
of
antioxidative
stress
and
in
Ames
dwarf
mice,
GH and
IGF
deficiency
may
cause
oxidative
stress
[9].
In
addition,
Ames
dwarf
mice
have
increased
level
of
adiponectin
and
reduced
expression
of
interleukin-6
(IL-6)
and
tumor
necrosis
factor-alpha
(TNF-a)
[10,
11].
Ames
dwarf
mice
with
pituitary
grafts
at
21
days
showed
increased
lympho-
cytes
in
the
spleen
and
thymus,
splenic
natural
killer
(NK)
cell
activity
and
peripheral
white
blood
cells
[12].
The
gastrointestinal
tract
is
exposed
to
a
large
variety
of
food
antigens
and
resided
with
a
huge
amount
of
commensal
bacteria,
playing
an
important
role
in
human
health.
[13].
The
intestinal
tract
is
also
an
active
player
of
the
local
and
systemic
immune
system,
participating
in
both
the
innate
and
adaptive
immune
responses
[14].
Impairment
of
intestinal
epithelial
barrier
[15]
and
environmental
factors
(like
gut
microbiota,
antibiotics
and
diet)
[16]
may
cause
immunological
imbalance
and
influence
distinct
arms
of
the
immune
response.
Impaired
gastrointestinal
function
contributes
to
aging
[18],
and
the
hypothalamic-
pituitary-adrenal
axis
modulates
the
gut
microbiota
in
mice
[19].
In
the
intestine,
GH
regulates
enteroendocrine
cell
secretion,
calcium
absorption,
and
intestinal
amino
acid
and
ion
transport.
GH
also
functions
in
the
growth
of
intestinal
mucosa
and
increase
the
proliferative
activity
of
intestinal
stem
cells
[20],
which
is
critical
to
the
gut
mucosal
integrity
and
immunity.
Therefore,
the
somatotropic
signaling-deficient
mice
provide
a
novel
model
for
investigation
of
the
role
of
GH
in
intestinal
development
and
immunity
[21-22].
To
date,
however,
the
mucosal
development
and
intestinal
immunity
of
the
Ames
dwarf
mice
remains
unclear.
In
this
current
study,
we
observed
the
colon
structure
and
the
inflammatory/
immune
cells
in
the
colon
lamina
propria
(cLP)
and
MLNs
in
2
months
(juvenile)
and
6
months
(adult)
old
Ames
dwarf
and
age-matched
WT
control
mice.
The
results
demonstrated
abnormal
cryptic
development
and
alterations
in
the
innate
immune
cells
(such
as
neutrophils,
monocytes,
eosinophils,
y8
T
cells,
CD11c+
DCs
and
CD11b+
macrophages)
and
adaptive
immune
cells
(i.e.,
B220+
B
cells,
Thl,
Th17
and
Treg
cells).
This
study
represents
the
first
observation
of
intestinal
immunity
in
Ames
dwarf
mice.
RESULTS
Dwarf
mice
demonstrate
age-related
alterations
in
colon
development
and
MLN
weight
Juvenile
and
adult
Ames
dwarf
mice
are
smaller
in
body
weight
and
body
length
than
age-matched
WT
mice
(Figure
1A),
which
is
consistent
with
literature
report
[23].
However,
the
colon
length
and
MLNs
weight
of
Ames
dwarf
mice
showed
age-related
changes.
The
colons
of
juvenile
Ames
dwarf
mice
were
ap-
proximately
40%
shorter
than
those
of
juvenile
WT
mice,
but
the
difference
was
reduced
to
about
24%
in
adult
Ames
dwarf
mice
(Figure
1B).
The
colon
length
of
WT
mice
was
not
changed
from
juvenile
to
adult.
These
data
indicate
that
colon
development
was
completed
by
two
months
old
in
WT
mice,
but
not
in
Ames
dwarf
mice.
A
great
age-related
change
was
observed
in
MLNs.
As
shown
in
Figure
1C,
MLNs
were
small
in
juvenile
Ames
dwarf
mice
with
a
ratio
of
MLNs
over
body
weight
at
an
average
of
0.00159
(n=5)
vs.
0.00229
in
juvenile
WT
mice.
In
adult
Ames
dwarf
mice,
MLNs
were
much
larger
with
a
ratio
of
MLNs
to
body
weight
at
an
average
of
0.00137
(n=5)
vs.
0.00086
in
adult
WT
mice.
These
gross
data
indicate
age-related
changes
in
colon
length
and
MLNs
size
in
Ames
dwarf
mice.
Shorter
colonic
crypts
in
Ames
dwarf
mice
The
gross
changes
of
colon
length
in
Ames
dwarf
mice
encouraged
an
evaluation
of
colon
histology.
Colonic
crypts
which
are
composed
of
epithelial
cells
contribute
to
host-microbial
homeostasis,
anti-
microbial
defense
and
modulation
of
immune
response
[22].
The
length
of
a
crypt
is defined
by
cell
number
in
the
crypt
and
varies
in
different
parts
of
colon.
We
evaluated
the
cell
number
in
the
crypts
of
proximal
colons
(PC)
and
distal
colons
(DC),
respectively.
The
results
showed
that
the
colonic
crypts
in
PC
and
DC
were
shorter
in
Ames
dwarf
mice
than
in
WT
mice.
In
juvenile
mice,
the
proximal
colon
had
15±3.5
cells/crypt
in
WT
mice,
but
only
11±2.3
cells/crypt
in
Ames
dwarf
mice
(n=30,
P
<
0.001);
the
distal
colon
had
20±2.7
cells/crypt
in
WT
mice
and
18±2.8
cells/crypt
in
dwarf
mice
(n=30,
P
<
0.001)
(Figure
2A).
In
adult
mice,
the
proximal
colon
had
16±2.7
cells/crypt
in
WT
mice,
but
only
12±2.9
cells/crypt
in
dwarf
mice
(n=30,
P
<
0.001);
the
distal
colon
had
22±4.4
cells/crypt
in
WT
mice
and
19±3.1
cells/crypt
in
dwarf
mice
(n=30,
P
<
0.001)
(Figure
2B).
These
data
indicate
that
the
Ames
dwarf
mice
have
developmental
abnormalities
in
colonic
crypts.
www.aging-us.com
359
AGING
***
I
I
01
P=0.052
* * *
40-
WT
dwarf
WT
dwarf
juvenile
adult
**
*
"2"
20
<-)
15
2
10
8
5
CO
2]
I
WT
dwarf
WT
dwarf
juvenile
adult
B
juvenile
adult
WT
dwarf
WT
dwarf
C
juvenile
adult
WT
dwarf
WT
dwarf
**
15
P=0.43
***
***
10
O
5-
U
WT
dwarf
WT
dwarf
0
0.002
**
*
7
0.001-
0
0
82
u
00u
juvenile
adult
P=0.14
***
0.003
tin
•-
A
WT
dwarf
WT
(kart'
juvenile
adult
Figure
1.
Age-related
alterations
in
body
weight,
colon
length
and
MLN
weight
of
dwarf
mice.
(A)
Body
weight
(left
panel)
and
body
length
(right
panel)
in
juvenile
and
adult
mice.
(B)
Colon
length
in
juvenile
(left
panel)
and
adult
(middle
panel)
mice.
Right
panel,
average
length
of
five
colons.
(C)
MLN
size
in
juvenile
(left
panel)
and
adult
(middle
panel)
mice.
Right
panel,
ratio
of
MLN
weight/body
weight
in
five
mice.
*,
P
<
0.05;
**,
P
<
0.01
and
***,
P
<
0.001
compared
to
WT.
Alterations
of
inflammatory
and
immune
cells
in
colonic
lamina
propria
(cLP)
of
Ames
dwarf
mice
Defects
in
colon
development
may
alter
the
intestinal
immune
cells.
We
observed
inflammatory
cells
in
colonic
lamina
propria
(cLP).
The
results
showed
that
the
mean
percentage
of
Ly6C+
Ly6G+
neutrophils
(A:
0.154
vs.
0.035
in
juvenile
and
0.089
vs.
0.027
in
adult)
and
Ly6C+
Ly6G-
monocyte
(A:
7.97
vs.
5.15
in
juve-
nile
and
6.67
vs.
3.79
in
adult)
was
noticeably
increased
in
the
cLP
of
juvenile
and
adult
Ames
dwarf
mice
when
compared
to
WT
mice
at
the
same
age
(Figure
3A),
indicating
mild
inflammation
in
the
colon
of
Ames
mice.
Interestingly,
the
percentage
of
eosinophils,
a
type
of
blood
cell
involved
in
allergic
reaction
and
parasitic
infection,
was
decreased
in
juvenile
(A:
0.72
vs.
1.61)
and
adult
(A:
3.14
vs.
5.06)
Ames
dwarf
mice
(Figure
3B).
www.aging-us.com
360
AGING
juvenile
WT
dwarf
)
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s
4144,1
1
1;',O'N:121
slek
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A
t
-
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-
4
ib
20-
15
•••.
10
5-
WT
dwarf
***
b.•
PC
25-
20-
,
C!A4o.
`'--
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s
4
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t
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l4
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s
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sz•
***
J
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DC
4.7-11.•
ti
p
dwarf
vr
r tc
4
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41
11,2-4.
.1w
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0;4
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1
-40',
,
4—
.
4:
S•40•-•
,;.N•
•*.•
WT
PC
3
.A
'
,
'
.:,
,
,.
,
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6
1.
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5
4
, i
.
'
,
.
le
i
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1
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4,_-4(.,-,
,
,
,:;
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?4,-
---
-
-
***
Cry
p
t
leng
t
h
(c
20
15
0
WT
dwarf
***
M.
.4.
r
r
r'
.vN
4s1
.:/e
t
.3
lic
N
r.
.)
1,:vvr,
41
\
A/4
A
WT
dwarf
B
adult
WT
dwarf
Figure
2.
Colonic
crypt
length
in
dwarf
mice.
H&E
staining
sections
of
proximal
colon
(PC)
and
distal
colon
(DC)
were
used
for
evaluation
of
colonic
crypt
length.
(A)
Juvenile
mice.
Left
panel,
images
of
colonic
crypts;
right
panel,
cryptic
cell
number
from
40
integrated
crypts
per
mouse.
(B)
Adult
mice.
Left
panel,
images
of
colonic
crypts;
right
panel,
cryptic
cell
number
from
40
integrated
crypts
per
mouse.
N=5;
***,
P
<
0.001
compared
to
WT.
Scare
bar:
50pm.
We
further
evaluated
antigen
presenting
cells
(APCs)
in
the
cLP
of
Ames
dwarf
mice,
including
CD11b+
macrophages,
CD1
lc+
dendritic
cells
(DCs)
and
y8
T
cells.
The
results
showed
that
the
mean
percentage
of
CD1
lb+
macrophages
(A:
3.22
vs.
0.81),
CD1
1
c+
DCs
(A:
1.39
vs.
0.78)
and
y8
T
cells
(A:
5.56
vs.
1.35),
was
www.aging-us.com
361
AGING
6.67
6
4
3
0.089
O
markedly
increased
in
juvenile
dwarf
mice
when
compared
to
WT
counterparts
(Figure
4A-C),
but
in
adult
dwarf
mice
only
DCs
(A:
0.864
vs.
0.689)
were
slightly
increased
(Figure
4B).
The
increased
APCs
encouraged
us
to
investigate
the
adaptive
immune
cells
in
cLP.
The
results
showed
that
it
was
mainly
the
Thl
helper
cells
(A:
2.6
vs.
1.4)
that
increased
in
juvenile
Ames
dwarf
mice
(Figure
5A),
but
was
mainly
the
Th17
helper
cells
(A:
8.3
vs.
4.7)
in
adult
dwarf
mice
(Figure
5B).
Treg
cells
were
not
noticeably
changed
in
both
juvenile
(A:
11.2
vs.
11.6)
and
adult
(A:
12.4
vs.
12.0)
Ames
dwarf
mice
(Figure
5C).
In
addition,
we
observed
a
decrease
of
B220+
B
cells
(A:
68.6
vs.
80.9)
in
juvenile
dwarf
mice,
but
not
in
adult
dwarf
mice
(Figure
5D).
Together
these
data
indicate
the
changes
of
intestinal
immunity
in
Ames
dwarf
mice.
Changes
of
immune
cells
in
mesenteric
lymph
nodes
of
Ames
dwarf
mice
Considering
the
changes
in
the
size
of
the
MLNs
and
in
immune
cells
of
cLP,
we
further
investigated
the
immune
cells
in
MLNs.
As
summarized
in
Table
1,
the
A
juvenile
WT
dwarf
percentage
of
CD1
1
c+
DCs
was
higher
in
both
juvenile
(A:
0.581
vs.
0.469)
and
adult
(A:
0.57
vs.
0.453)
dwarf
mice
when
compared
to
WT
mice.
However,
the
CD11b+
macrophages
showed
age-related
variations.
The
CD11b+
macrophages
were
less
in
the
juvenile
dwarf
mice
(A:
0.46
vs.
1.38),
but
more
in
adult
dwarf
mice
(A:
3.47
vs.
1.35)
compared
with
WT
mice.
The
percentage
of
y8
T
cells
was
slightly
higher
at
(A:
0.903
vs.
0.817)
in
juvenile
dwarf
mice,
but
is
lower
at
(A:
1.45
vs.
1.76)
in
the
adult
dwarf
mice.
Furthermore,
we
observed
less
Thl
cells
in
juvenile
(A:
0.171
vs.
0.373)
and
adult
(A:
1.29
vs.2.15)
and
B
cells
in
juvenile
(A:
33.4
vs.
38.3)
and
adult
(A:
32.4
vs.
37.2).
However,
the
percentage
of
Th17
cells were
greatly
increased
at
(A:
0.627
vs.
0.093)
in
juvenile
dwarf
mice
while
no
differences
were
observed
in
adult
dwarf
mice.
These
data
indicate
age-related
changes
of
immune
cells
in
MLNs.
DISCUSSION
The
present
study
investigated
the
colon
development
and
immune
cells
in
the
cLP
and
MLNs
of
Ames
dwarf
adult
WT
dwarf
7
5
10
4
2
,
0
6
5
Mono
4ytes
0.154
0.027
3.79
Neutrophils
0.035
5.15
7.97
O
0
1
10
2
103 104 10 106 107
10
10
1
10
2
10
3
0
4
10
6
07
10
10
1
10
2
10
3
104
1
0
0
6
10
7
10
8
10
1
10
2
0
3
10
10
10
6
.7
10
Ly6C
B
juvenile
adult
WT
dwarf
WT
dwarf
7
Eosinophils
1.61
0
6
5
3
2
0.72
10
6
4
10
5.06
10
6
3
2
3.14
0
7
3
2
10
1
10
2
10
3
0
10 10
6
10
7
7
10
1
0
2
10
3
0
4
10
10
6
10
0
2
0
3
10
7
10
1
10
0
10
CD45
Figure
3.
Inflammatory
cells
in
the
cLP
of
dwarf
mice.
(A)
Neutrophils
and
monocytes
in
juvenile
and
adult
mice.
(B)
Eosinophils
in
juvenile
and
adult
mice.
Data
(percentage)
in
images
indicate
the
results
from
a
pool
of
cLP
cells
from
5
mice.
www.aging-us.com
362
AGING
mice
with
WT
mice
at
the
same
age
as
a
control.
Ames
dwarf
mice
are
GH-deficient
due
to
a
mutation
in
a
pituitary-specific,
paired-like
homeodomain
transcript-
tion
factor
gene,
Propl.
The
growth
and
maturation
of
dwarf
mice
are
delayed
and
we
assayed
the
immune
cells
at
the
ages
of
2
months
and
six
months
old,
respectively.
Herein
we
termed
the
mice
at
2
months
as
juvenile
and
mice
at
6
months
as
adult.
Ames
dwarf
mice
were
40-50%
less
of
body
weight
compared
to
their
WT
counterparts
at
the
same
ages,
but
the
ratio
of
MLNs
to
body
weight
was
dramatically
changed
with
age.
In
juvenile
dwarf
mice,
the
MLN
to
body
weight
was
smaller
in
Ames
dwarf
mice
than
in
WT
mice,
but
in
adult
dwarf
mice
this
ratio
was
larger,
A
juvenile
WT
dwarf
indicating
an
active
immune
response
in
the
intestine
of
adult
dwarf
mice.
The
colons
were
significantly
shorter
in
juvenile
and
adult
dwarf
mice
than
in
WT
mice,
but
when
compared
between
the
juvenile
and
adult
Ames
mice,
the
colons
of
adult
dwarf
mice
were
longer,
indicating
the
delayed
maturation
of
colon
in
the
Ames
dwarf
mice.
More
significantly,
the
dwarf
mice
had
shorter
crypt
length
in
both
the
proximal
colon
and
distal
colon
when
compared
with
WT
counterparts,
indicating
that
the
deficiency
of
somatotropic
signaling
(GH/
IGF-1),
together
with
TSH,
THs
and
prolactin
signaling,
may
affect
the
growth
and
development
of
the
colonic
crypts.
Dwarf
mice
have
a
delay
in
puberty,
and
it
is
largely
due
to
lack
of
thyroid
hormone
[24].
adult
WT
dwarf
rn
bn
0.807
BOOK
600K
400K
200K
1M
800K
600K
400K
200K
1M
800K
500K
400K
200K
0.657
3.22
ODOK
500K
4DOK
200K
0.684
7
0
10 10
10
10
10
2
10
10
10
10
6
10
7
10
1
10
2
10
3
10
0
10
10
7
10
10
2
10
10
10
10
6
10
7
CD1lb
11A
BOOK
600K
400K
200K
B
1M
BOOK
500K
400K
200K
U
dD
to
DCs
0.782
1M
800K
600K
400K
200K
1.39
0.689
800K
600K
400K
200K
0.864
10
1
10
2
10
7
10
1
10
2
10
10 10
10
6
10
7
0
2
10
10
10
10
10
8
0
1
10
2
10
0
10
10
6
10
7
o.
CD11c
C
7
E
L2
1
?—
2
1.35
ryS
T
I
5.56
1.58
4
1
10
6
5
1.65
10
5
3
10
1
0
3
10
10
1
10
2
10
3
10
10
10
6
10
7
10
1
10
2
0
3
10
10 10
6
10
7
10
1
10
2
10
10
6
10
7
al
10
10
1
0
10
7
CD3
Figure
4.
Antigen
presenting
cells
in
the
cLP
of
dwarf
mice.
(A)
CD11b+
macrophage,
(B)
CD11c+
dendritic
cells
and
(C)
y6
T
cells
in
the
cLP
of
juvenile
and
adult
mice.
Data
(percentage)
in
images
indicate
the
results
from
a
pool
of
cLP
cells
from
5
mice.
www.aging-us.com
363
AGING
Table
1.
Innate
and
adaptive
immune
cells
in
MLNs
of
juvenile
and
adult
Ames
dwarf
mice.
Values
are
mean
percentage
(A).
Cells
juvenile
adult
Gating
WT
(%)
dwarf
(%)
WT
(%)
dwarf
(%)
Tregs
10.5
7.7
10.9
11.5
FoxP3+
(FSC-SSClow
7AAD-
CD4+)
Thl
0.373
0.171
2.15
1.29
IFNy+
(FSC-SSClow
7AAD-
CD4+)
Th17
0.093
0.627
0.74
0.63
IL17+
(FSC-SSClow
7AAD-
CD4+)
y6
T
0.817
0.903
1.76
1.45
y6TCR+
(FSC-SSClow
7AAD-
CD3+)
Macrophages
1.38
0.46
1.35
3.47
CD11b+
(FSC-SSClow
7AAD-
CD45+
)
Dendritic
cells
0.469
0.581
0.453
0.571
CD11c+
(FSC-SSClow
7AAD-
CD45+
)
B
cells
38.8
33.4
37.2
32.4
B220+
(FSC-SSClow
7AAD-
CD45+)
Herein
we
found
the
developmental
defects
of
the colon
in
Ames
dwarf
mice,
but
it
is
unknown
how
the
deficiency
of
somatotropic
signaling
affects
the
development
of
the
colon.
Further
study
is
warranted.
Intestinal
epithelial
cells
(IECs)
could
produce
various
types
of
cytokines
and
chemokines,
which
function
as
immunoregulatory
signals
for
directing
immune
cell
response
against
foreign
antigens
[25].
The
defect
of
colonic
crypts
in
Ames
dwarf
mice
may
dysfunction
the
immuno-regulatory
signaling
and
lead
to
alterations
of
the
innate
and
adaptive
immune
response.
In
addition,
IECs
are
a
single
layer
of
cells
that
separate
the
host
from
gut
contents
that
are
enriched
with
pathogens
and
commensal
bacteria.
The
intestinal
microbiota
plays
a
crucial
role
in
the
development
of
local
and
systemic
immunity
[26-27].
For
example,
colonization
of
the
small
intestine
of
mice
with
segmented
filamentous
bacteria
could
induce
Th17
cells
response
and
antimicrobial
defense
[16]
and
increase
the
number
of
Treg
cells
in
the
small
intestine
and
colon
[28].
Colonization
of
animals
with
the
gut
microorganism
Bacteroides
fragilis
directs
the
cellular
and physical
maturation
of
the
developing
immune
system,
promot-
ing
Thl
/Th2
balance
[29].
Additionally,
microbiota
could
also
drive
the
expansion
of
B
and
T
cells
in
Peyer's
patches
and
mesenteric
lymph
nodes
[30],
promoting
IgA
secretion
[31].
Therefore,
we
further
investigated
the
inflammatory
and
immune
cells
in
the
cLP
and
MLNs.
Neutrophils
and
monocytes
are
the
first-responders
of
inflammatory
cells
that
migrate
towards
the
site
of
in-
flammation,
and
play
specific
and
nonspecific
defensive
functions.
In
both
juvenile
and
adult
dwarf
mice,
neutrophils
and
monocytes
were
increased
in
the
cLP
when
compared
to
WT
mice,
indicating
presence
of
mild
inflammation
in
the
colon
of
dwarf
mice.
Interes-
tingly,
the
eosinophils
were
decreased
in
juvenile
and
adults
dwarf
mice
compared
to
WT
mice.
Eosinophils
are
innate
immune
cells
that
function
in
regulation
of
inflammation,
epithelial
barrier,
tissue
remodeling
and
bridging
of
innate
and
adaptive
immunity
[32].
Eosinophil
peroxidase
forms
reactive
oxygen
species
and
reactive
nitrogen
intermediates
that
can
promote
oxidative
stress
and
killing
of
microbial
pathogens.
The
decreased
eosinophils
in
the
colon
suggest
the
dys-
function
of
intestinal
barrier
and
inflammation.
The
increased
inflammatory
cells
in
cLP
were
accompanied
with
the
elevation
of
antigen
presenting
cells
in
juvenile
and
dwarf
mice,
including
CD11b+
macrophage,
CD11c+
DCs
and
y8
T
cells.
The
y8
T
cells
are
innate-like
lymphoid
cells
and
can
function
in
the
resolution
of
infection
by
multiple
ways,
such
as
TCR-MHCII
independent
antigen
presentation
and
recruitment
of
effector
cells
like
neutrophils
and
macrophages,
playing
an
important
role
in
the
immune
surveillance
[33].
The
increase
of
these
cell
populations
suggests
enhanced
pathogen
exposure
of
the
colon
in
dwarf
mice
with
defects
in
cryptic
development,
which
is
consistent
with
the
presence
of
mild
inflammation
as
indicated
by
increased
inflammatory
cells.
However,
it
is
noteworthy
that
hematopoietic
stem
cells
(HSCs)
are
increased
in
juvenile
and
adult
Ames
dwarf
mice
[34].
Whether
the
changes
of
HSC
in
bone
marrow
influence
the
inflammatory
cells
in
cLP
of
Ames
dwarf
mice
is
warranted
for
further
study.
www.aging-us.com
364
AGING
68.6
0
1
10 2
10
10
7
74.6
10
1
10
2
0
3
10
0
6
10
BOOK
600K
400K
200K
80.9
10
1
10
2
10
0
10
0
6
10
7
800K
600K
400K
200K
BOOK
600K
400K
200K
BOOK
600K
400K
200K
71.8
0
1
10
2
10
3
10
10
10
6
le
10
7
7
WT
juvenile
dwarf
adult
le
6
WT
dwarf
7
io
6
105_
1.4
5
2.6
3.8
2.4
10
4
,
4
10
4
0
3
cj
10
2
:
10
2
10
2
101
10
10
2
10
10
2
10
3
10 10
10
10
1
10
2
10
2
10
10 10
10
f0
• •
I
74
•:4
f0
10
10
7
fi
10
6
4.7
8.3
1.9
2.0
10
I
4
4
2
10
2
0
1
10
2
0
3
10
10
6
0
7
o
2
0
10
0
f0
10
2
10
3
10
10
10
1
10
2
10
3
1
0
10
7
10
7
10
6
10
6
:
10
6
10
8
11.2
10
6
:
11.6
10
5
:
12.4
12.0
4
10
:
10
10
3
:
10
2
10
2
:
10
1
10
1
10
1
10
2
10
3
10
0
10
6
10
7
'
5
'
;O
r
;O W
"
10
10
10 10
10
1010
2
le
10
10
10
6
le
le
E
4
CD4
A
B
C
D
B220
Figure
5.
Adaptive
immune
cells
in
the
cLP
of
dwarf
mice.
(A)
Th1
cells,
(B)
Th17
cells,
(C)
Treg
cells
and
(D)
B
cells
in
the
cLP
of
juvenile
and
adult
mice.
Data
(percentage)
in
images
indicate
the
results
from
a
pool
of
cLP
cells
from
5
mice.
Furthermore,
consistent
with
increased
antigen
presenting
cells,
adaptive
immune
cells
were
also
increased
in
dwarf
mice
although
variations
occurred
in
juvenile
and
adult
dwarf
mice.
Thl
cells
were
increased
in
juvenile
dwarf
mice,
but
slightly
decreased
in
adult
dwarf
mice.
Th17
cells
were
not
altered
in
juvenile
dwarf
mice,
but
markedly
increased
in
adult
dwarf
mice.
Literature
data
show
the
pivotal
beneficial
role
of
IL-17A
for
the
integrity
of
the
intestinal
epithelial
barrier,
and
mice
with
deficiency
of
IL-17
display
a
broad
vulnerability
to
various
infectious
pathogens
[35].
The
increased
Th17
cells
in
adult
dwarf
mice
may
play
a
protective
role,
contributing
to
host
defense
and
barrier
integrity
in
the
dwarf
mice.
The
abnormal
size
of
MLNs
(ratio
of
MLN/body
weight)
indicates
increased
inflammatory/immune
responses.
Thus
we
investigated
the
antigen
presenting
cells
and
adaptive
immune
cells
in
MLNs.
Results
showed
a
decrease
of
CD1
lb+
macrophages,
Thl
and
Treg
cells,
but
a
great
increase
of
Th17
cells
in
juvenile
dwarf
mice.
In
the
adult
dwarf
mice,
CD11b+
macrophages
were
elevated
about
3
fold.
These
results
indicate
an
active
immune
response
of
MLNs
to
intestinal
pathogens.
www.aging-us.com
365
AGING
Colonization
of
gut
microbiota
in
early
life
plays
an
instrumental
role
in
the
development
and
education
of
the
host
immune
system
[36],
and
alterations
of
intestinal
commensals
have
profound
effects
on
the
structural
and
functional
development
of
the
immune
system,
such
as
T
cell
response
[37].
This
study
found
that
in
Ames
dwarf
mice,
the
deficiency
of
GH,
PRL
and
TSH
led
to
defects
of
colonic
epithelial
proliferation
and
cryptic
development
although
the
underlying
mechanism
is
unclear
yet.
As
an
important
immune
tissue
and
barrier,
this
may
cause
abnormal
immune
response,
commensal
colonization
and
bacterial
infiltration.
Our
results
in
inflammatory
and
immune
cells
in
the
cLP
and
MLNs
support
this
hypothesis
and
are
of
important
significance.
The
Ames
dwarf
mice
may
be
a
great
novel
model
to
understand
intestinal
homeostasis,
commensal
colonization
and
immune
development.
MATERIALS
AND
METHODS
Animals
Male
Ames
dwarf
mice
were
produced
in
our
closed
breeding
colony
at
Southern
Illinois
University
School
of
Medicine
(SIUSOM)
by
breeding
homozygous
mutant
males
(PropT')
with
heterozygous
females
(Propl
+/-
).
All
breeding
pairs
avoided
brother
x
sister
mating
to
ensure
genetic
diversity.
Heterozygous
normal
(WT)
animals
were
used
as
controls
for
the
homozygous
mutant
dwarfs.
Animals
entered
the
study
at
2
and
6
months
of
age.
All
animals
were
housed
and
bred
in
a
temperature-
controlled
(22°C)
room
with
a
daily
photoperiod
of
12
hr:12
hr
as
light:dark.
Rodent
food
(Formulab
Laboratory
Diet,
PMI
Nutrition
International,
Inc.,
St.
Louis,
MO)
and
tap
water
were
supplied
ad
libitum.
All
animal
procedures
were
approved
by
the
SIUSOM
Animal
Care
and
Use
Committee.
H&E
staining
Antibodies
and
flow
cytometry
intestinal
contents
with
cold
PBS,
the
colons
from
5
mice
were
opened
longitudinally,
pooled
and
cut
into
small
pieces
of
—1
cm
in
length,
followed
by
incubation
twice
for
20
mM
in
PBS
supplemented
with
2%
FBS,
5
mM
EDTA
and
1mM
D,L-dithiothreitol
(DTT;
American
Bioanalytical).
The
tissues
were
then
cut
into
1
mm
pieces
and
further
incubated
in
HBSS
(Hank's
Balanced
Salt
Solution)
in
the
presence
of
0.5
mg/nil
collagenase
D
(Roche),
0.5
mg/nil
dispase
II
(Roche)
and
100
U
DNase
I
(Sigma)
for
two
consecutive
20
min
at
37°C.
After
digestion,
cells
were
passed
through
a
40i.tm
nylon
cell
strainer
(Fisher
Scientific)
and
then
recovered
by
Percoll
gradient
centrifugation
at
2500
rpm
for
20
min.
Leukocytes
were
recovered
at
the
interface
of
40%/80%
Percoll,
then
washed
and
kept
in
cell
staining
buffer
(BioLegend).
For
cells
isolated
from
the
MLN,
cut
the
tissue
into
pieces,
then
pass
the
tissue
through
a
401.tm
nylon
cell
strainer
(Fisher
Scientific)
by
using
a
plunger,
the
cells were
collected,
washed
and
then
kept
in
cell
staining
buffer
(BioLegend).
Cell
stimulation
and
staining
For
flow
cytometry
analysis,
isolated
cells
from
the
cLP
and
MLNs
were
pre-incubated
with
an
Fc
receptor-
blocking
mAb
(CD16/32;
2.4G2)
for
10
minutes
at
4
°C,
then
incubated
with
saturating
amounts
of
FITC-,
PE-
and
APC-conjugated
mAbs
for
30
minutes
at
4
°C.
To
assess
intracellular
IL-17A,
IL-22
and
IFNy,
cells
were
stimulated
for
5hr
in
medium
RPMI
1640
containing
10%
FBS,
50
ng/ml
Phorbol
12-Myristate
13-Acetate
(PMA;
SigmaAldrich,
St.
Louis,
MO)
and
500
ng/ml
ionomycin
(Sigma-Aldrich)
in
the
presence
of
GolgiStop
(BD
Pharmingen).
After
surface
staining,
stimulated
cells
were
fixed
with
fixation
buffer
(BioLegend)
and
permeabilized
with
permeabilization
wash
buffer
(BioLegend),
followed
by
intracellular
cytokine
staining.
To
detect
intracellular
Foxp3,
a
BioLegend
true-nuclear
transcription
factor
buffer
set
was
used
for
fixation
and
permeabilization
of
the
cells.
Hematoxylin
and
Eosin
(H&E)
staining
was
performed
using
a
standard
laboratory
protocol.
In
short,
tissues
were
fixed
overnight
in
10%
formaldehyde
for
paraffin
embedding.
The
embedded
samples
were
sectioned
at
4i.tm
thickness,
followed
by
staining
with
H&E
staining.
Sections
were
examined
using
400x
magnification,
all
images
were
captured
on
a
microscope
(Olympus,
Japan).
Crypt
length
was
defined
as
cell
number
per
crypt
by
counting
30
integrated
crypts
per
mouse.
Cell
isolation
Colonic
lamina
propria
cells
were
isolated
following
an
established
protocol
[38].
Briefly,
after
flushing
the
Abs
for
FACS
analysis,
Fc
y
receptor-blocking
mAb
(CD16/32;
2.4G2),
FITC-conjugated-conjugated
mAbs:
CD3
(145-2C11),
IFN-y
(XMG1.2),
CD45
(30-F11);
PE-conjugated
mAbs:
TCRy8
(GL-3),
CD4
(GK1.5),
Ly6G
(1A8),
CD1
1
c
(N418),
B220
(RA3-6B2),
Siglec-
F(
E50-2440);
APC-conjugated
mAbs:
IL17
(TC11-
18H10.1),
CD1
lb
(M1/70),
and
Ly6C
(HK1.4)
were
used.
The
florescence
of
the
cells
was
analyzed
using
an
Accuri
C6
flow
cytometer
(Accuri,
Ann
Arbor,
MI,
USA)
and
the
data
were
analyzed
with
FlowJo
software
(TreeStar,
San
Carlos,
CA).
Cell
gating
strategies
are
shown
in
Figure
S
1.
Briefly,
after
excluding
the
debris
and
doublets,
cells
were
gated
on
7AAD-live
cells,
then
www.aging-us.com
366
AGING
CD45+
leukocytes.
All
the
percentages
of
immune
cells
in
the
paper
were
the
mean
percentage
(n=5)
indicated
as
A.
Statistical
analyses
Statistical
analyses
were
carried
out
with
Prism
4
(Graph
Pad
software,
CA).
Variance
test,
Student
t
test,
or
one-way
ANOVA
test,
as
appropriate,
were
used
to
compare
the
difference
between
WT
and
Ames
dwarf
mice
with
p
<
0.05
as
statistical
significance.
*,
P
<
0.05,
**,
P
<
0.01
and
***,
P
<
0.001
compared
with
WT.
ACKNOWLEDGEMENTS
We
would
like
to
thank
Dr.
Minglin
Lin
and
Dr.
Yimin
(Julia)
Fang
for
helping
with
mice
dissection.
CONFLICTS
OF
INTEREST
Authors
declare
that
there
are
no
conflicts
of
interest.
FUNDING
This
work
was
supported
by
National
Institutes
of
Health
(NIH)
grants
(R21AG051869
and
R01AG019899
to
AB)
and
National Natural
Science
Foundation
of
China
(81772842).
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369
AGING
SUPPLEMENTARY
MATERIAL
Scatter
FSC-singlets
SSC-singlets
I
3M
iM
1M
BOOK
600K
400K
200K
A
nn
BOOK
600K
400K
200K
1M
3M
5M
7M
FSC-A
1M
3M
5M
7M
1
10
FSC-A
200K
400K
600K
800K
1M
SSC-A
Live
CD45+
1M
A
1M
800K
600K
400K
200K
BOOK
600K
400K
200K
0
1
10
2
0
10
4
10
10
6
10
7
10
1
10
2
10
3
10
4
10
10
7AAD
CD45
Supplemental
Figure
1.
Gating
strategies
in
the
analysis
of
leukocytes
from
cLP.
From
left
to
right:
Scatter
gates
to
exclude
debris,
singlets
gates
to
exclude
doublets,
live
gate
to
exclude
7AAD
+
dead
cells,
and
then
gate
on
CD45+
leukocytes.
FSC,
forward
light
scatter;
SSC,
side
light
scatter.
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370
AGING