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The University ofTexas Health Science Center at Houston and the Texas Heart Institute at
St. Luke s Episcopal Hospital, Houston, Texas, USA
Received June 29, 2012 - Accepted January 2013
The first two authors contributed equally to this study
In patients with obesity and diabetes mellitus, abnormal production of inflammatory factors may
result in cardiovascular dysfunction. In the current study, we tested the impact of CDld-mediated
innate immune responses on the expression and activation of NFKB in the hearts of adipose diabetic
(db/db) mice. Splenocytes from adult db/db and CDld-knockout mice of both genders and their wildtype, C57BL/6 and Balb/C counterparts were examined for tumor necrosis factor (TNF)-a and TNF-a
receptor type 1. The percentage of natural killer T (NKT) cells in CD3+ T cells was compared with
that in nondiabetic control mice. Despite the absence of inflammatory infiltrates, the hearts of db/db
mice showed alterations in TNF-a receptor-I and NFKB activity, including increased expression of both
the NFKB p52 and p65 subunits. In the hearts of CDld-knockout mice, p52 expression was reduced,
while p65 expression remained largely unchanged. On echocardiography, the ratio of E to A transmitral
flow velocities (an indicator of diastolic function) was significantly decreased in db/db mice after they
swam for 30 minutes. These results provide evidence for CDld-mediated NFKB activation and diastolic
dysfunction in the hearts of db/db mice. Therefore, CDld-associated abnormalities of innate immune
responses and TNF-a production in splenic tissue may contribute to NFKB activation and cardiac
dysfunction in type 2 diabetes.
and interleukin-l ~, chronic hyperglycemia creates a
proinflammatory milieu that leads to insulin resistance
and cardiovascular inflammatory complications,
such as atherosclerosis, ischemic myocardial disease,
heart failure, and dilated cardiomyopathy (2).
Researchers are increasingly recognizing that
activation of the innate immune system plays
a role in the development and progression of
insulin resistance by inducing chronic, low-grade
Key words: obesity, diabetes mellitus, type 2, nuclear transcription factor CDld, diastolic dysfunction, low-grade
chronic inflammation
Mailing address: Yong-Jian Geng, MD, PhD,
Professor and Director, Center for Cardiovascular Biology
and Atherosclerosis Research, Cardiology Division,
Department ofIntemal Medicine, The University of Texas
Medical School at Houston, 6431 Fannin Street,
MSB 1.246, Houston, TX 77030, USA.
Tel.: +1713 5006073/713 5006569 Fax: +1 713 5006556
e-mail: yong-jian.geng@uth.trnc.edu
0394-6320 (2013)
copyright by BIOLlFE, s.a.s.
This publication and/or article is for individual use only and may not be further
reproduced without written permission from t1iecopyright holder.
Unauthorized reproduction may result in financial and other penalties
59
60
R. MADONNA ET AL.
Materials
All chemicals were purchased from Sigma-Aldrich (St.
Louis, Missouri, USA) unless otherwise specified. Type-I
collagenase was obtained from Worthington Biochemical
Corporation (Lakewood, New Jersey, USA).
Blood collection and biochemical assays
Blood was drawn via a cardiac puncture, collected into
nonheparinized tubes, allowed to clot at room temperature
for 1 hour, and centrifuged for 20 minutes at 2,000 x g.
The serum was removed and stored at -20 DC until assays
were performed. Levels of glucose, triglyceride, and
the total, high-density lipoprotein (HDL], low-density
lipoprotein (LDL), and very-low-density lipoprotein
(VLDL) cholesterol were measured by standard clinical
chemistry laboratory methods (Equine Laboratories,
Houston, Texas, USA).
Flow cytometry
After being anesthetized with an intraperitoneal
injection of 65 mg/kg ketamine and 13 mg/mL xylazine
in sterile normal saline, the wild-type C57BL/6 and db/db/
C57BL/6 mice were euthanized. The spleens of male db/
db mice and age- and sex-matched C57BL/6J control mice
were removed, mashed by the plunger of the syringe, and
passed through a 70 IlM cell strainer. After red-blood-cell
removal, spleen cells were plated in RPMI 1640 culture
medium supplemented with 20% fetal bovine serum
(Gibco, Invitrogen; Life Technologies, Grand Island, New
York, USA), penicillin (100 U/mL), and streptomycin
sulfate (100 mg/mL). For flow cytometry analysis and cell
sorting, spleen cells (1x 106) were incubated for 30 minutes
at 4DC with 10 ul, ofphycoerythrin-conjugated mouse antiCDld antibody (Santa Cruz Biotechnology, Santa Cruz,
California, USA). An antibody specific for the FcyRIII/II
receptor (BD Biosciences, San Jose, California, USA) was
used to inhibit nonspecific staining. Cells were analyzed
with a BS LSRII flow cytometer (BD Biosciences). For
each sample, 30,000 events were acquired and analyzed
with CellQuest software (BD Biosciences). Subsequently,
61
62
R. MADONNA ET AL.
RESULTS
C57
(J)
(J)
db/db
~ b
.j a
J
-c 3
55.2%
D...
(J)
(J)
"j
1
~
CD1d-PE
80
60
63
.' .
i
.;~
'00
(J)
<1> 40
....
a.
x
;.
<1>
"0
......
CD1d-PE
0
U
20
0
C57
D
C57
db/db
.....
db/db
*
1.6
1.4
> 0:::: 1.2
~ E
1.0
"O
.-~ ......
roo 0.8
Qi U
0.6
0::::
0.4
0.0
0
CD1d
GAPDH
QiZ
C57
db/db
Fig. 1. CDld expression in spleens from C57BL/6J and db/db mice. Panels A, B) Flow cytometry analysis of CDld
exp ression in spleen cells from male db/db mice, compared with the respective sex- and age-matched (12-month-old) wildtype C57BL/6J control mice. In Panel A, the scatter plots are representative of 3 independent experiments. Controls for
flow cytometry included an IgG isotype control antibody. In Panel B, the columns and bars represent the mean percentage
standard deviation (SD) ofCDld-positive cells (*P<O.05, db/db mice versus C57BL/6J control mice) . Panels C, D)
Quantitative real-time polymerase chain reaction (qRT-PCR) assessment ofCDld mRNA exp ression in spleen cells from
male db/db mice, compared with their resp ective sex- and age-matched (12-month-old) wild-type C57BL/6J control mice.
The columns and bars represent the mean values SD from 3 independent experiments assessing mRNA expression after
normalization with GAPDH. In Panel C. agarose gel [electrophoresis?] ofqRT-PCR [products?] is representative of3
independent experiments (*P<O.05, db/db mice versus C57BL/6J control mice).
64
R. MADONNA ET AL.
C57
db/db
TNF a
GAPDH
B
7
z
0::: 5
0
-z 5
~o:::
~ E 4
**
**
z
Gi..- 4
> ....
~.8 3
QJ Co
> QJ
,." o
QJ 0
~ u.3
.!1 z
~1-2
db/db
>
QJ 0
-QJ
~
Co
QJ
.!1
LL
0:::,
I-
C57
E 6
Gi N
> ....
0:::
~ ~
QJ 0
0::: ,
QJ 0
LL
I-
C57
db/db
C57
db/db
Fig. 2. Expression oftumor necrosis factor (TNF)-a and TNF-a receptorl in spleens and hearts from C57BLl6J and db/db
mice. Panels A, B) Quantitative real-time polymerase chain reaction (qRT-PCR) assessment ofTNF-a mRNA expression
in spleen cells from male db/db mice, compared with the respective sex- and age-matched (l2-month-old) wild-type
C57BLl6J control mice. In Panel A, agarose gel electrophoresis ofqRT-PCR products is representative of 3 independent
experiments. Panel B, the columns and bars represent the mean values standard deviation (SD) from 3 experiments
assessing mRNA expression after normalization with GAPDH (**P<O.Ol, db/db mice versus C57BLl6J control mice).
Panel C) qRT-PCR assessment ofTNF-a receptor1 mRNA expression in hearts from male db/db mice, compared with the
respective sex- and age-matched (l2-month-old) wild-type C57BLl6J control mice. The columns and bars represent the
mean values SD from 3 experiments assessing mRNA expression after normalization with GAPDH (**P<O.Ol, db/db
mice versus C57BLl6J control mice).
C57
M
db/db
F
Balb/C
CD1d-l -
DC
..
65
-I
p65
c
Ant i-p65
C57
M
F
+ - + -
[e65
Lamin B
I Lamin B
db/db
M
+
F
- + -
123456789
-
supershift
Fig. 3. NFKB expression and activity in hearts from db/db and nondiabetic control mice. Panel A) Levels of the p65
subunit ofNFKB protein in nuclear protein extracts isolatedfrom 12-month-old db/db male (M) andfemale (F) mice and
sex-matched nondiabetic C57BL/6 control mice. Panel B) Levels of the p65 subunit ofNFKB protein in cytosolic and
nuclear protein extracts isolatedfrom 12-month-old male CD1d-knockout and sex-age matched Balb/C control mice. The
blots are representative of 3 independent experiments. Similar results were obtainedfrom female mice. Panel C) NFKB
activity was determined by an electrophoretic mobility shift assay, performed by mixing a 32P-labeled oligonucleotide
that codes for the consensus sequence ofNFKB binding promoter with nuclear proteins from the hearts ofmale (M) and
female (F) db/db mice and sex- and age-matched (l2-month-old) C57BL/6 control mice (3 mice per group). Specificity of
NFKB binding activity was evaluated by cold oligonucleotide analysis and supershifting with anti- NFKB antibody. The
electrophoretic run with nuclear protein extracts from C57BL/6 control hearts is shown in lanes 1-4. The electrophoretic
run with nuclear protein extracts from db/db mice is shown in lanes 5-8. The autoradiogram is representative 3 separate
gel shift experiments for NFKB. Lane 9 shows the electrophoretic runs with protein extracts omitted or with cardiac
nuclear extracts incubated with a cold probe.
66
R. MADONNA ET AL.
Cytosol
0.;
U ~
130
96
Total
,.,.
0.;
() ~
o0
p100
130 -
96-
72
43-
34
...
Heart
100
C
**
CD
C
80
0
o
c
p52
43 -
200
>
~
0..
C57
db/db
100
80
O
w 60
...
(5
0..
CD
80
>
~
40
40
~ 20
N
LO
p52
B-actin
LO
p100
I
o
.~
72-
2c
C
160
0
...
>
~
~ 20
'):
Heart
**
CD
0..
CD
Q:j
~-actin
(5
40
()
.;
34-
O
w 120
...
0..
130 96-
o
c
] 60
0
0..
CD
p100
Splee n
C
,,0
55-
--
B-actin
Cytosol
'):
cl (]
34-
-I
()
...
55 -
Total
se
,.
72-
p52
55
43
Total
LO
0..
C57
db/db
Balb/C CD1d-l -
Fig. 4. RelB/p52 expression and activity in hearts and spleens from CDId-knockout and db/db mice. Levels of the
endogenous p l 00 precursor and p52 active subunit ofNF-kB complex in total and cytosolic heart protein extracts isolated
from male db/db mice (Panel A) and male CDId knock-out mice (Panel C), compared with the respective background
sex- and age-matched (l2-month-old) wild-type C57BL/6 and Balb/C control mice. Panel B) Levels ofthe endogenous
plOOprecursor and p52 active subunit ofNF-kB complex in total spleen protein extracts isolatedfrom male db/db mice
compared with sex- and age-matched (l2-month-old) wild-type C57BL/6 control mice. The blots are representative of3
independent experiments. The results ofscanning densitometry (3 mice per group) are expressed as arbitrary units. The
columns and bars represent the mean values standard deviation (**P<O.OI, db/db mice versus control mice).
Balb/C
db/db
C57
Balb/ C
CD1-1
11 11 11 11
TC
COl -I-
II
~ g i b"'gi
<? .<f ~ <?.<f ~
blood
TCTC
67
TC
130 -
130
96
plOD
72
55
p5 2
43
plOD
96
72 55
p52
43
34
34
_~
I~-actin
~ - actin
Fig. 5. RelBlp52 expression and activity in lymph nodes, spleen and bloodfrom CDId-knockout and BalblC control mice.
Panel A) Levels ofpIOOlp52 expression in total and cytosolic protein extracts isolated from blood of male dbldb mice
and CDI d knock-out mice compared with sex- and age-matched wild-type BalblC and C57BL/6 controls mice. Panel B)
Levels of endogenous pI 00 precursor and p52 active subunit ofNF-KB complex in total protein extracts isolatedfrom
spleen, lymph nodes and blood of male CDI d knock-out mice comp ared with sex- and age-matched wild-typ e BalblC
controls. Blots are representative ofthree independent experiments.
68
R. MADONNA ET AL.
ORO
H&E
FITC-sarc a-actinin
Sirius red
Fig. 6. Histological analyses of heart sections from nondiabetic controls and db/db mice. Representative Oil Red a
stained cross-sections of left ventricular myocardium from male db/db (A) and sex- and age-matched (l2-month-old)
C57BL/6 control mice (E), showing accumulation oflipids in db/db cardiac myocytes. Hearts were harvested, embedded
in optimal-cutting-temperature compound, and counterstained with hematoxylin and eosin (H&E) (3 hearts per group).
Similar results were observed in female mice. Panels Band F: Representative H&E-stained cross-sections of left
ventricular myocardium from male db/db mice (B) and sex- and age-matched (l2-month-old) C57BL/6 control mice
(F). Similar results were observed in female mice. Panels C and G: Representative immunofluorescence-stained crosssections ofleft ventricular myocardiumfrom male db/db (C) and sex- and age-matched (l2-month-old) C57BL/6 mice (G)
stained for sarcomeric a-actin in. Similar results were observed in female mice. Panels D and H) Representative Sirius
red-stained cross-sections ofleft ventricular myocardium from male db/db (D) and sex- and age-matched (l2-month-old)
C57BL/6 mice (H). Similar results were observed infemale mice. Magnification: LO>.
DISCUSSION
An association between low-grade chronic
inflammation and cardiovascular complications in
insulin resistance and type 2 diabetes is increasingly
being recognized (l). However, the molecular and
cellular basis of immune dysregulation leading
to low-grade chronic inflammation and cardiac
malfunction in type 2 diabetes has not been fully
elucidated. Our current study provides experimental
evidence that a heightened innate immune response
occurs through a CD 1d-mediated mechanism .
In the spleen, CD 1d-restricted, lipid-responding
NKT cells result in a variety of immune reactions,
db /db
pre-swimming
o
~-r
- E
III
0-
(1)-
>
69
db /db
post-swimming
~-l
o
III
0-
-(1)E
>
Fig. 7. Echocardiographic index ofdiastolicfu nction in nondiabetic controls and db/db mice. Panels A, B) Representative
Doppler flow recordings fro m db/db mice at baseline (A) and after swimming (B), showing decreased peak E-wave
heights in db/db mice and impaired left ventricular relaxation after exercise. Panel C) The E/A ratio (measured as the
ratio between the peak height ofthe mitral flo w E-wave and that of the A-wave on Doppler flo w recording) in db/db and
C57 mice at baseline and after swimming. Each dot indicates the mean value derived fr om at least 3 measurements in
each individual mouse. The lines indicate mean values in each group ofmice (n=7).
R. MADONNA ET AL.
70
NK-T Cell
Cardiac
myocyte
Dendritic Cell
Dysfunction
(jJ
TCR
Glycolipid
CD1d
Fig. 8. CDId-associated expression of NFKB and cardiac dysfunction in type 2 diabetes. In type 2 diabetes, TNF-a
production in splenic tissue may contribute to NFKB activation and cardiac dysfunction. NKT: natural killer T; TNF:
tumor necrosis factor; TNFR: tumor necrosis factor receptor; TCR: T-cell receptor.
71
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R. MADONNA ET AL.
6.
7.
8.
9.
10.
11.
12.
13.
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