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Vascular Medicine
Immune Activation Resulting From NKG2D/Ligand
Interaction Promotes Atherosclerosis
Mingcan Xia, MD, MS; Nadia Guerra, PhD; Galina K. Sukhova, PhD; Kangkang Yang, PhD;
Carla K. Miller, RD, PhD; Guo-Ping Shi, DSc; David H. Raulet, PhD; Na Xiong, PhD
Background—The interplay between the immune system and abnormal metabolic conditions sustains and propagates a
vicious feedback cycle of chronic inflammation and metabolic dysfunction that is critical for atherosclerotic progression.
It is well established that abnormal metabolic conditions, such as dyslipidemia and hyperglycemia, cause various
cellular stress responses that induce tissue inflammation and immune cell activation, which in turn exacerbate the
metabolic dysfunction. However, molecular events linking these processes are not well understood.
Methods and Results—Tissues and organs of humans and mice with hyperglycemia and hyperlipidemia were examined
for expression of ligands for NKG2D, a potent immune-activating receptor expressed by several types of immune cells,
and the role of NKG2D in atherosclerosis and metabolic diseases was probed with the use of mice lacking NKG2D or
by blocking NKG2D with monoclonal antibodies. NKG2D ligands were upregulated in multiple organs, particularly
atherosclerotic aortas and inflamed livers. Ligand upregulation was induced in vitro by abnormal metabolites associated
with metabolic dysfunctions. Using apolipoprotein E– deficient mouse models, we demonstrated that preventing
NKG2D functions resulted in a dramatic reduction in plaque formation, suppressed systemic and organ inflammation
mediated by multiple immune cell types, and alleviated abnormal metabolic conditions.
Conclusions—The NKG2D/ligand interaction is a critical molecular link in the vicious cycle of chronic inflammation and
metabolic dysfunction that promotes atherosclerosis and might be a useful target for therapeutic intervention in the
disease. (Circulation. 2011;124:2933-2943.)
Key Words: atherosclerosis 䡲 immune system 䡲 inflammation 䡲 liver 䡲 NKG2D
A
long with other risk factors, such as metabolic dysfunction, immune cells are critically involved in promoting
atherosclerosis.1,2 Abnormal metabolic conditions induce
stress responses in cells of artery walls to initiate vascular
inflammation and result in the recruitment of immune cells
that enhance the inflammation and promote plaque formation.
Conversely, chronic inflammation exacerbates metabolic dysfunction, creating a vicious cycle of chronic inflammation
and metabolic dysfunction that propagates atherosclerosis.3
Editorial see p 2809
Clinical Perspective on p 2943
Macrophages are the most abundant immune cells in
plaques,1 where they accumulate lipid contents and secrete
proinflammatory cytokines and chemokines. Various lymphocytes, including T and natural killer (NK) cells, are also
detected in plaques and involved in atherosclerosis.4 – 6 A role
for T/B cells in atherosclerosis was initially suggested by the
findings that chow-fed Rag1⫺/⫺apolipoprotein E– deficient
(ApoE⫺/⫺) mice, which lack T/B cells, developed atherosclerotic lesions more slowly than did ApoE⫺/⫺ control mice.7
Subsequent experiments suggested a role for CD4⫹ T cells in
promoting the disease by responding to antigens associated
with abnormal metabolites and enhancing production of
interferon-␥ (IFN-␥).8 Additional studies found a modest
decrease in plaque formation in mice lacking NKT cells, a
T-cell subset reactive against specific lipids.6,9 On the other
hand, regulatory T cells inhibit atherosclerosis by suppressing
immune activation.10 Innate lymphocytes such as NK cells
have also been implicated in promoting atherosclerosis.6,11
Involvement of immune cells in atherosclerosis is thought
to be initiated by abnormal metabolic conditions and the
Received March 29, 2011; accepted September 27, 2011.
From the Center of Molecular Immunology and Infectious Diseases, Departments of Veterinary and Biomedical Sciences (M.X., K.Y., N.X.) and
Nutrition (C.K.M.), Pennsylvania State University, University Park; Department of Molecular and Cell Biology, University of California, Berkeley (N.G.,
D.H.R.); and Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA (G.K.S., G.S.). Current address for
N.G. is Division of Cell and Molecular Biology, Imperial College, London, UK. Current address for C.K.M. is Department of Human Nutrition, Ohio
State University, Columbus.
Guest Editor for this article was Donald D. Heistad, MD.
The online-only Data Supplement is available with this article at http://circ.ahajournals.org/lookup/suppl/doi:10.1161/CIRCULATIONAHA.
111.034850/-/DC1.
Correspondence to Na Xiong, PhD, Department of Veterinary and Biomedical Sciences, Pennsylvania State University, 115 Henning Bldg, University
Park, PA 16802. E-mail [email protected]
© 2011 American Heart Association, Inc.
Circulation is available at http://circ.ahajournals.org
DOI: 10.1161/CIRCULATIONAHA.111.034850
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December 20/27, 2011
resulting stress to arterial endothelial cells. Abnormal metabolic components activate endothelial cells and macrophages
of susceptible regions such as aortic arches through Toll-like
receptors 2/4 to promote vascular inflammation and atherosclerosis.12,13 Stressed endothelial cells also upregulate
chemokines and adhesion molecules (including CCL5,
CXCL1, macrophage migration inhibitory receptor,
E-selectin, and vascular cell adhesion molecule 1) to recruit
atherogenic immune cells that promote vascular inflammation (reviewed in Rao et al14). Immune cells, including NK
and NKT cells, are also activated in other inflamed organs,
such as the liver, to contribute indirectly to the atherosclerosis
by increasing systemic inflammation and metabolic dysfunction. However, molecular events that link activation of
different immune cells and abnormal metabolic conditions in
atherosclerotic progression are not well understood.
NKG2D (also called killer cell lectin-like receptor subfamily K member 1 or KLRK1) is a potent activating receptor
expressed by several types of immune cells involved in
atherosclerosis, including NK, NKT, ␥␦ T, and subsets of
activated ␣␤ T cells.15,16 Multiple different membrane proteins have been identified as ligands for NKG2D. In humans,
they include 2 major histocompatibility complex class I
chain–related family members (MICA and MICB) and 5
UL16-binding protein family members (ULBP1-4 and
RAET1G). In mice, they include 5 retinoic acid early transcripts 1 family members (Rae-1␣, ␤, ␥, ␦, and ⑀), 3
histocompatibility 60 family members (H60a, H60b, and
H60c), and MULT-1 (UL16-binding protein-like transcript
1).15 These ligands are generally expressed at low levels or
not at all on healthy cells but are upregulated under various
pathological conditions such as tumorigenesis, viral infection,
and genotoxic stress. Interaction of the ligands with NKG2D
stimulates or costimulates the NKG2D-expressing immune
cells to proliferate, produce cytokines (including IFN-␥,
granulocyte-macrophage colony-stimulating factor, macrophage inflammatory protein 1-␣, and interleukin-2), and/or
lyse target cells that express the ligands.15–20 Although the
upregulation of NKG2D ligands plays an important role in
host defense against tumors and viral infections,19,21 their
abnormal upregulation could contribute to immune-mediated
inflammatory diseases such as arthritis, celiac disease, and
type 1 diabetes mellitus.22–25 We report herein that the
NKG2D ligands were upregulated in humans and mice with
hyperglycemia and/or hyperlipidemia and play a critical role
in atherosclerosis, liver inflammation, and associated metabolic disease.
Methods
Mouse Models and Human Samples
ApoE⫺/⫺ mice on the C56BL/6 background were purchased from
Jackson Laboratory. NKG2D knockout mice (Klrk1⫺/⫺ mice) on the
C56BL/6 background were described recently.26 ApoE⫺/⫺ and
Klrk1⫺/⫺ mice were intercrossed to generate Klrk1⫺/⫺ApoE⫺/⫺
mice. The Klrk1 and ApoE knockout genotypes were determined by
genomic polymerase chain reaction, as exemplified in Figure I in the
online-only Data Supplement.
In the Western diet (WD)–accelerated model of atherosclerosis,
male ApoE⫺/⫺ and Klrk1⫺/⫺ApoE⫺/⫺ mice were switched from
normal chow to a high-fat diet (35.5% Fat, Bio Serv) at the age of 6
weeks and analyzed for plaques, blood cytokines, and metabolites 8
to 9 weeks later. In the streptozotocin-induced diabetic model of
atherosclerosis,27 6-week-old male ApoE⫺/⫺ and Klrk1⫺/⫺ApoE⫺/⫺
mice were injected intraperitoneally with streptozotocin (55 mg/kg in
0.05 mol/L citrate buffer, pH 4.5) daily for 6 days. The treated mice
were confirmed diabetic (blood glucose ⬎350 mg/dL) and fed on
chow for an additional 8 to 9 weeks before analysis. In both models,
the Klrk1⫺/⫺ApoE⫺/⫺ and ApoE⫺/⫺ mice were always treated and
analyzed in parallel.
In experiments to test the effect of NKG2D antibody blockade on
atherosclerosis, 6-week-old male ApoE⫺/⫺ mice were injected intraperitoneally with monoclonal anti-NKG2D (clone MI-6, rat IgG2a)16
or isotype-matched control antibodies 1 week and 3 days before the
streptozotocin treatment and weekly afterward for 8 weeks (200 ␮g
for the first injection and 100 ␮g for subsequent injections). The
mice were analyzed 8 to 9 weeks after the beginning of streptozotocin treatment. All procedures were approved by the Institutional
Animal Care and Use Committee of Pennsylvania State University.
Use of biosamples of humans was approved by the institutional
review boards of Pennsylvania State University and Harvard Medical
School.
Antibodies and Flow Cytometry
The NKG2D antibody (MI-6) was prepared in house. Pan-Rae-1
antibody was purchased from R&D Systems. PE-anti-CD31, PECy5CD11b, PECY7-anti-CD3, PE-anti-TCR␤, PE-anti-F4/80, and
Alexa647-anti-NKG2D antibodies were purchased from eBioscience. FITC-anti-NK1.1, PECy5-anti-TCR ␤ , Alexa488-antiinterleukin [IL]-6, PE-anti-IL-4, and PECY7-anti-IFN-␥ antibodies
and the mouse IL-6 enzyme-linked immunosorbent assay set were
from BD Bioscience. For flow cytometry, cells were stained with the
proper combination of antibodies (indicated in the figure) and
analyzed on a flow cytometer FC500 (Beckman Coulter).
En Face Oil Red O Staining and Quantification of
Plaque Formation
Aortas beginning at the root outside the heart and stopping at the
thoracic diaphragm were collected, opened up longitudinally, fixed
in 10% formalin, and stained with Oil red O dye as described.12 Sizes
of total aortic arch and neighboring regions and of Oil red O–staining
plaque areas within the regions were calculated on the basis of digital
pictures of the stained aortas with the use of Adobe Photoshop, as
reported.28 The extent of plaque formation was expressed as the
percentage of Oil red O–stained area of the total area of the aortic
arch and neighboring region.
Immunohistochemical Staining
Cryosections of mouse atherosclerotic plaques were stained with
polyclonal goat anti-mouse Rae-1 or H60 antibody (R&D Systems or
Santa Cruz Biotechnology, respectively). Cryosections of human
aortic plaques were stained with monoclonal anti-MICA/B (clone
6D4, eBioscience), anti-CD68, or anti-CD31 antibodies, performed
in a manner similar to that described previously.29
Cellular Isolation
Mononucleocytes, endothelial cells, and hepatocytes were isolated
from aortas or livers according to published procedures.30,31 Briefly,
the liver was perfused with 3 mL 50 U/mL heparin (Sigma-Aldrich)
and 3 mL 0.05% collagenase A/0.05% Dispase II (Roche) via the
portal vein and then pressed through a cell strainer (PGC Scientific).
Liver mononucleocytes and hepatocytes were separated by 40%/80%
Percoll solution (Sigma-Aldrich). For isolation of cells from normal
aortas and atherosclerotic aortic plaques, mouse aortas were first
perfused with 50 U/mL heparin and then digested with an enzyme
cocktail dissolved in phosphate-buffered saline (125 U/mL collagenase type XI, 60 U/mL hyaluronidase type I-s, 60 U/mL DNase1,
450 U/mL collagenase type I, and 20 mmol/L Hepes, Sigma-Aldrich)
at 37°C for 1 hour. The isolated cells were counted and used for
analyses.
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NKG2D Promotes Atherosclerosis
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Figure 1. Upregulation of NKG2D ligands in sera and aortic plaques of patients with type 2 diabetes mellitus. A, A high percentage of
patients with type 2 diabetes mellitus have elevated levels of soluble major histocompatibility complex class 1–related chain A (MICA) in
the sera. The dashed line indicates the detection limit. One dot represents 1 sample, and the short flat lines indicate average concentrations. Fisher exact test was used to compare proportions of healthy vs diabetic people with detectable MICA expression. *P⬍0.05,
**P⬍0.01, ***P⬍0.001 (applied for all figures). B through J, Detection of MICA/B proteins by immunohistochemical staining on human
aortic plaques. Cryosections of the plaques were stained with anti-MICA/B antibodies (B, D, F, H), whereas adjacent sections were
stained with anti-CD68 or CD31 antibodies to reveal macrophages or endothelial cells (C, E, G, I). A section of normal aorta was used
as a control (J).
Real-Time Reverse Transcription Polymerase
Chain Reaction Analysis
RNA was analyzed for transcripts of Rae-1␦, Rae-1⑀, and H60b by
quantitative real-time reverse transcription polymerase chain reaction, as described previously.18,19,32
In Vitro Treatment of Macrophages
Resident cells were flushed out of the peritoneal cavity of naive
C56BL/6 mice with phosphate-buffered saline and plate-adhered
overnight. The adherent macrophages were cultured in 10% fetal
bovine serum–supplemented Dulbecco’s modified Eagle’s medium
containing 10 ␮g/mL human oxidized low-density lipoprotein (LDL)
(medium-oxidized with copper [II] sulfate), natural LDL (Kalen
Biomedical), 200 ␮g/mL advanced glycation end products (Fitzgerald Industries International), or lipopolysaccharides for 2 days33 and
then analyzed for expression of NKG2D ligands.
Western Blot Analysis of Total Rae-1 Proteins
The macrophages were lysed, separated on polyacrylamide gel
electrophoresis gels, transferred to polyvinylidene fluoride membrane, blotted with anti-Rae-1 antibody (C20, Santa Cruz Biotechnology), and developed with an enhanced chemiluminescence system (GE Healthcare Life Sciences).
Multiplex Analysis of Serum Cytokines
Mouse sera were analyzed directly on a mouse proinflammatory7plex kit (IL-6, tumor necrosis factor-␣, IFN-␥, IL-12p70, IL-10,
IL-1␤, and KC/CXCL1) (Meso Scale Discovery, Gaithersburg, MD).
Assessment of Cholesterol and Triglyceride Levels
in Serum
Levels of cholesterol and triglycerides were analyzed on a Vet Test
Chemistry Analyzer (IDEXX Laboratories, Inc, Maine).
Alanine Aminotransferase Activity Analysis
Alanine aminotransferase activities in the serum were determined
with the use of a kit from Bioo Scientific (Austin, TX) according to
the manufacturer’s instruction.
Ex Vivo Culture of Liver Explants
Phosphate-buffered saline–perfused livers were excised from WDfed ApoE⫺/⫺ or Klrk1⫺/⫺ApoE⫺/⫺ mice. Equal weights of excised
livers were cut into ⬇1-mm3 cubes and cultured in media for 1 or 3
days. The culture media were recovered and analyzed for cytokines
by enzyme-linked immunosorbent assay.
Intracellular Cytokine Staining
Mononucleocytes isolated from livers were cultured in media overnight in the presence of brofeldin A and analyzed by intracellular
cytokine staining for the production of IL-6 and IFN-␥ in gated
subsets of immune cells according to the manufacturers’ instructions
(eBioscience and Biolegend).
Enzyme-Linked Immunosorbent Assay Detection
of Soluble MICA
MICA proteins in sera were assayed with the use of an enzymelinked immunosorbent assay kit (R&D Systems, Minneapolis, MN).
Statistical Analyses
Data are expressed as mean⫾SE. Two-tailed Student t tests were
used to determine statistical significance for 2-group comparisons.
The ANOVA test was used for analysis of combined results of
repeated experiments or multiple-group comparison (with Tukey
adjustment). The Fisher exact test was used to compare proportions
of diabetic and healthy people with detectable levels of soluble
MICA proteins (sMICA) in sera. P⬍0.05 is considered significant.
Results
Upregulation of NKG2D Ligands in Sera and
Atherosclerotic Plaques of Humans With
Metabolic Dysfunction
To investigate whether NKG2D or its ligands are involved in
atherosclerosis associated with metabolic dysfunction, we
first sought evidence that NKG2D ligands were upregulated
in patients with type 2 diabetes mellitus (Table I in the
online-only Data Supplement) by assessing sMICA in their
sera. sMICA are enzymatically cleaved products of membrane MICA and were detected abundantly in patients with
various cancers and immune disorders, but not healthy
people.22,34,35 Of 22 patients with diabetes mellitus tested,
45% (10) had detectable sMICA (⬎25 pg/mL), and 27% (6)
had high levels (400 –12250 pg/mL), whereas in the healthy
controls, only 11% (2/19) had detectable levels, and 5%
(1/19) had high levels of sMICA in sera (Figure 1A). These
results suggest that a high percentage of patients with type 2
diabetes mellitus have upregulated MICA expression. We
tried to associate the sMICA detection in the patients with
other parameters (ages, years with diabetes mellitus, and
glucose levels) but found no correlation (Table I in the
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Figure 2. Preferential upregulation of NKG2D ligands in atherosclerotic aortas and livers of apolipoprotein E– deficient (ApoE⫺/⫺) mice.
A, Real-time reverse transcription polymerase chain reaction analysis of Rae-1␦, Rae-1⑀, and H60b transcripts in RNA samples isolated
from aortic arch regions of Western diet (WD)–fed ApoE⫺/⫺ mice. Samples of age-matched WD- and chow-fed wild-type (WT) mice
were used as controls. The experiment included 3 mice per group and was representative of 2 repeated experiments of a total of 6
mice. B, Immunohistochemical staining of sections of atherosclerotic aortic arch regions (top and middle) and “plaque-free” thoracic
regions (bottom) of the same WD-fed ApoE⫺/⫺ mice with Rae-1 (left) or H60 antibodies (right). The pictures in the middle are higher
amplifications (⫻400) of the boxed areas of the top panels (⫻100). C, Flow cytometry of cell surface Rae-1 expression on macrophages (gated on the CD45⫹CD11b⫹F4/80⫹ population) and endothelial cells (CD45⫺CD31⫹ population) isolated from atherosclerotic
aortas of WD-fed ApoE⫺/⫺ mice or normal aortas of WT mice. The gray areas indicated isotype-matched control antibody staining. D,
Real-time reverse transcription polymerase chain reaction analysis of Rae-1␦ transcripts in indicated organs of WD-fed ApoE⫺/⫺ mice
and WT mice. ***Statistical significance compared with values of corresponding tissues of WT mice; n⫽4. E, Real-time reverse transcription polymerase chain reaction analysis of Rae-1␦ in purified macrophages of livers of WD-fed ApoE⫺/⫺ mice and WT mice; n⫽2.
F and G, Flow cytometry of Rae-1 expression on CD11b⫹F4/80⫹ liver macrophages (F) and hepatocytes (G) of WD-fed ApoE⫺/⫺ mice.
The experiment included 3 to 4 mice per group and was performed twice with similar results. H, Flow cytometry of Rae-1 expression
on peritoneal macrophages of WT mice cultured in vitro in presence of oxidized low-density lipoprotein (OxLDL) and advanced glycation end products (AGE) as in C except that the dashed lines depict staining in the presence of a 10-fold excess of unlabeled anti-panRae-1 antibodies. The experiment was performed 3 times, with pools of cells from 3 mice for each, with similar results. I, Western blot
analysis of Rae-1 proteins in macrophages cultured in vitro in presence of oxidized low-density lipoprotein or natural low-density lipoprotein (nLDL). The lipopolysaccharide (LPS) treatment was included as a positive control.33 The blot was performed 3 times with similar results.
online-only Data Supplement), suggesting that these factors
might not be directly associated with the MICA upregulation.
We also performed immunohistochemical staining of atherosclerotic aortic sections of humans to assess expression of
NKG2D ligands in the tissues. In contrast to plaque-free
aortas (Figure 1J), atherosclerotic aortas contained many cells
that stained positive with an anti-MICA/B antibody (Figure
1B, 1D, 1F, and 1H). The overlapping staining patterns of
MICA/B with CD68 or CD31 in adjacent sections suggested
that both macrophages and endothelial cells of the atheroscle-
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Figure 3. NKG2D plays a critical role in
plaque formation in Western diet (WD)–
fed and streptozotocin (STZ)-diabetic
apolipoprotein E– deficient (ApoE⫺/⫺)
mice. A, Visibly different plaque sizes in
aortic arch regions of representative
WD-fed ApoE⫺/⫺ and Klrk1⫺/⫺ApoE⫺/⫺
mice. The whitish plaque areas are indicated by arrows. The pictures shown are
representative of 16 to 18 mice of each
genotype. B, En face Oil red O staining
to detect lipid-deposited plaque areas in
aortas of representative ApoE⫺/⫺ (n⫽12)
and Klrk1⫺/⫺ApoE⫺/⫺ (n⫽10) mice.
Plaque areas stain red. The regions analyzed for percentage of plaque are
boxed. C and D, Quantitative comparison of percentages of average Oil
red O staining–positive plaque areas
in the total aortic arch regions of
Klrk1⫺/⫺ApoE⫺/⫺ and ApoE⫺/⫺ mice that
were fed on a WD (C) or treated with
streptozotocin (D). E, Quantitative comparison of percentages of average Oil
red O staining–positive plaque areas in
streptozotocin-induced diabetic ApoE⫺/⫺
mice treated with NKG2D or control antibodies (Ab). Total numbers of mice analyzed in C, D, and E are indicated in the
graphs, which are compiled from data of
2 to 4 experiments for each model.
rotic aortas expressed MICA/B (Figure 1B through 1I). The
high expression of NKG2D ligands in aortic plaques suggested the potential involvement in atherosclerosis of immune activation mediated by NKG2D/ligand interactions.
Preferential Upregulation of NKG2D Ligands in
Tissue and Organs of ApoEⴚ/ⴚ Mice Where
Abnormal Metabolites Accumulate
To understand the NKG2D ligand upregulation process and
its role in atherosclerosis, we used the ApoE⫺/⫺ mouse model
of atherosclerosis. ApoE⫺/⫺ mice are defective in lipid
metabolism and develop plaques when they age, particularly
in susceptible regions such as aortic arch areas, and the
disease is markedly exacerbated when the mice are fed the
high-fat WD.
We first assessed the amounts of transcripts for NKG2D
ligands in RNA isolated from atherosclerotic aortic arch
regions of WD-fed ApoE⫺/⫺ mice. Compared with those of
either WD- or chow-fed wild-type (WT) C57BL/6 mice,
samples from WD-fed ApoE⫺/⫺ mice had much higher
(10 –30-fold) amounts of transcripts for Rae-1␦, Rae-1⑀, and
H60b, which are mouse ligands for NKG2D (Figure 2A).
These results suggest that the ligands are upregulated in the
atherosclerotic aortas. Consistent with this conclusion, the
atherosclerotic aortic arch regions of older chow-fed
ApoE⫺/⫺ mice also had much higher amounts of transcripts
for the NKG2D ligands than the atherosclerosis-resistant
thoracic aortic region of the same mice (Figure II in the
online-only Data Supplement).
Immunohistochemical staining with Rae-1 and H60 antibodies confirmed the preferential expression of NKG2D
ligands in plaques. Extensive Rae-1 and H60 staining was
detected within plaques of WD-fed ApoE⫺/⫺ mice of the
atherosclerotic aortic arch regions (Figure 2B, top and middle
panels). In contrast, Rae-1 or H60 staining was nearly
undetectable in plaque-free aortic regions of the same mice
(Figure 2B, bottom). To determine the types of cells that
express the ligands, we performed flow cytometric analysis of
cells isolated from atherosclerotic aortas of WD-fed ApoE⫺/⫺
mice. In contrast to those of normal aortas, many macrophages and endothelial cells of the atherosclerotic aortas had
appreciable cell surface Rae-1 expression (Figure 2C), consistent with the findings in humans.
Because metabolic dysfunctions are systemic conditions,
we tested whether other organs in the WD-fed ApoE⫺/⫺ mice
had upregulated NKG2D ligands. Rae-1 transcripts were
elevated dramatically in the liver and somewhat less so in the
kidney, whereas other organs tested showed lower elevations
(Figure 2D). Similar to the results with macrophages from
aortic plaques, liver macrophages of ApoE⫺/⫺ mice contained
significantly more Rae-1 transcripts than WT controls (Figure
2E), and cell surface expression of Rae-1 was also significantly, although modestly, increased (Figure 2F). Rae-1 was
also markedly increased on the surface of hepatocytes of the
ApoE⫺/⫺ mice, although WT hepatocytes exhibited a higher
basal level of Rae-1 than did liver macrophages (Figure 2G).
With the consideration that abnormal metabolites accumulate in both atherosclerotic plaques and livers, they may play
a role in the induction of NKG2D ligands. Indeed, WT
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Figure 4. Preventing NKG2D/ligand
interactions reduces serum levels of
inflammation-associated cytokines in
Western diet (WD)–fed and streptozotocin (STZ)-diabetic apolipoprotein E– deficient (ApoE⫺/⫺) mice. A, Serum levels of
multiple cytokines in WD-fed ApoE⫺/⫺
and Klrk1⫺/⫺ApoE⫺/⫺ mice; n⫽12 each.
B, Serum levels of multiple cytokines in
streptozotocin-diabetic ApoE⫺/⫺ and
Klrk1⫺/⫺ApoE⫺/⫺ mice; n⫽4 each. C,
Serum levels of multiple cytokines of
NKG2D antibody (Ab)–treated (n⫽9) and
control antibody–treated (n⫽6)
streptozotocin-diabetic ApoE⫺/⫺ mice.
Note that scales for different cytokines in
the different models are not the same. IL
indicates interleukin; KC, Chemokine
(C-X-C motif) ligand 1 (CXCL1); IFN,
interferon; TNF, tumor necrosis factor;
and BD, below detection limit.
macrophages could be reproducibly induced to upregulate
NKG2D ligands when cultured in vitro in the presence of
oxidized LDL or advanced glycation end products, 2 abnormal metabolites associated with dyslipidemia and hyperglycemia (Figure 2H and 2I and Figure III in the online-only
Data Supplement). The Rae-1 staining was competitively
inhibited by unlabeled Rae-1 antibodies, confirming the
specificity of the staining (Figure 2H). Together, these results
suggest that abnormal metabolic conditions induce upregulation of NKG2D ligands in various tissues and organs,
particularly those where abnormal metabolites accumulate,
such as aortic plaques and livers. When it is considered that
immune cell subsets such as NK and NKT cells in those
tissues express NKG2D (Figure IV in the online-only Data
Supplement) and promote atherosclerosis,6,9,11 the NKG2D/
ligand axis might play an important role in promoting
atherosclerosis through activation of these or other NKG2Dexpressing cells.
Preventing NKG2D/Ligand Interactions
Suppresses Plaque Formation in ApoEⴚ/ⴚ Mice
To directly determine the role of NKG2D in atherosclerosis,
we tested whether interfering with NKG2D function suppresses plaque formation in ApoE⫺/⫺ mice. Compared with
WD-fed ApoE⫺/⫺ mice, similarly fed NKG2D-deficient
Klrk1⫺/⫺ApoE⫺/⫺ littermates exhibited dramatically smaller
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Table. Metabolic Parameters of NKG2D Knockout and Anti-NKG2D Antibody–Treated ApoEⴚ/ⴚ Mice in Western Diet and
Streptozotocin Models of Atherosclerosis
Western Diet
ApoE⫺/⫺
(n⫽6)
Streptozotocin-ApoE⫺/⫺⫹Antibody
Streptozotocin
ApoE⫺/⫺Klrk1⫺/⫺
(n⫽6)
P
ApoE⫺/⫺
(n⫽6)
ApoE⫺/⫺Klrk1⫺/⫺
(n⫽6)
P
Control Antibody
(n⫽5)
NKG2D Antibody
(n⫽4)
P
Cholesterol, mg/dL
706⫾89
337⫾48
0.005
1284⫾339‡
1446⫾402
0.78
1537⫾528
1001⫾198
0.38
Triglycerides, mg/dL
98.3⫾7.6
57.5⫾6.8
0.002
85.0⫾8.0
57.5⫾13.5
0.22
84.8⫾16.3
81.5⫾19.7
0.9
Glucose, mg,dL
372⫾27*
308⫾14†
0.03
852⫾202§
550⫾85
0.3
794⫾94
578⫾53
0.1
*Fourteen samples were analyzed.
†Twelve samples were analyzed.
‡P⬍0.05 compared with the cholesterol concentrations of Western diet–fed apolipoprotein E– deficient (ApoE⫺/⫺) mice.
§P⬍0.001 compared with the glucose concentrations of Western diet–fed ApoE⫺/⫺ mice.
plaques in the aortas (Figure 3A through 3C). On average, the
percentage of lipid-deposited plaque area in the total aortic
arch region, as assessed by en face Oil red O staining, was
reduced 5- to 6-fold in Klrk1⫺/⫺ApoE⫺/⫺ mice (Figure 3B
and 3C). Oil red O staining of cross sections of aortic roots
also revealed significantly reduced plaque formation in
Klrk1⫺/⫺ApoE⫺/⫺ mice compared with the ApoE⫺/⫺ controls, but the effect was less dramatic than seen in the aortic
arch regions (Figure VA in the online-only Data Supplement). The different effects of NKG2D knockout on the
plaque reduction in the aortic arch region and aortic roots are
consistent with other reports showing that immune-mediated
mechanisms differentially influence plaque formation in different anatomic sites.36,37
To test whether the NKG2D/ligand axis is broadly involved in atherosclerosis that accompanies diabetic conditions, we also examined streptozotocin-treated ApoE⫺/⫺
mice. Because it is directly toxic for insulin-secreting
pancreatic ␤-cells, streptozotocin causes severe hyperglycemia and hyperlipidemia in mice by a mechanism similar to
that of type 1 diabetes mellitus.27 As in the WD-fed model,
streptozotocin-treated Klrk1⫺/⫺ApoE⫺/⫺ mice had much
smaller plaque sizes than similarly treated ApoE⫺/⫺ mice in
aortic arch regions (Figure 3D). The NKG2D knockout also
significantly, although less dramatically, reduced the amount
of plaque in the aortic root cross sections in the
streptozotocin-diabetic ApoE⫺/⫺ mice (Figure VB in the
online-only Data Supplement).
As an additional approach to test the role of NKG2D in
atherosclerosis, which might be also relevant for therapeutic
applications, we tested whether the induction of atherosclerosis in streptozotocin-diabetic ApoE⫺/⫺ mice was inhibited
by injection of NKG2D antibodies that are known to block
interactions of NKG2D with its ligands.16,24 Compared with
ApoE⫺/⫺ mice injected with control antibodies, those that
received the NKG2D antibodies had markedly reduced
plaque areas (Figure 3E). Together, these results demonstrate
a critical role of the NKG2D/ligand axis in atherosclerosis
induced under various abnormal metabolic conditions.
Preventing NKG2D/Ligand Interactions Reduces
the Systemic Production of Multiple Cytokine
Markers Associated With Immune Activation
Because the NKG2D/ligand interaction activates NKG2Dexpressing immune cells, which produce inflammatory cyto-
kines, preventing the interaction likely suppresses atherosclerosis by inhibiting immune activation. In support of this
conclusion, compared with ApoE ⫺/⫺ mice, NKG2Dknockout or NKG2D antibody–treated ApoE⫺/⫺ mice exhibited significant reductions in the amounts of multiple cytokines in the serum, including inflammatory cytokines (IL-6,
IFN-␥) and an immune regulatory cytokine (IL-10) (Figure
4). IL-6 and IFN-␥ are known to be involved in atherosclerosis38 and were markedly reduced in all of the models in
which NKG2D function was prevented, suggesting that
NKG2D/ligand-mediated immune cell activation associated
with atherosclerotic progression might function at least partly
through the increased production of these cytokines. IL-12
was also reduced in all of the models, although the difference
was not significant in the streptozotocin model. The other
cytokines studied showed more variable results.
NKG2D-Mediated Immune Activation Aggravates
Abnormal Metabolic Conditions by Promoting
Liver Inflammation and Dysfunction
Preventing NKG2D interactions also considerably alleviated
the abnormal metabolic conditions in the WD-fed ApoE⫺/⫺
model. Notably, the sera of WD-fed Klrk1⫺/⫺ApoE⫺/⫺ mice
were much less cloudy than the sera of similarly fed ApoE⫺/⫺
mice (Figure VI in the online-only Data Supplement), suggesting reduced lipid content. Direct analysis showed that
cholesterol and triglycerides were significantly reduced in the
Klrk1⫺/⫺ApoE⫺/⫺ mice (Table). The glucose levels were also
significantly, although modestly, reduced in the WD-fed
Klrk1⫺/⫺ApoE⫺/⫺ mice (Table).
Compared with WD-fed ApoE⫺/⫺ mice, streptozotocintreated ApoE⫺/⫺ mice had more severe hyperglycemia and
hyperlipidemia, neither of which were significantly corrected
by interfering with NKG2D (Table). These findings suggest
that the severe metabolic dysfunctions in the streptozotocin
model are not dependent on NKG2D-mediated immune
activation, consistent with the notion that streptozotocin
induces diabetes mellitus through its direct toxic effect on
pancreatic ␤-cells and other cells.39 Nevertheless, interfering
with NKG2D in the streptozotocin model suppressed production of the cytokines associated with immune activation and
atherosclerosis (Figure 3D), suggesting that preventing
NKG2D-mediated inflammation reduces atherosclerosis
without the requirements of alleviating overall metabolic
dysfunctions. However, in the WD model, the role of
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2940
Circulation
December 20/27, 2011
ligands in the ApoE⫺/⫺ mice, we tested whether the NKG2D/
ligand axis contributes to liver inflammation that results in
liver dysfunction. Compared with WD-fed ApoE⫺/⫺ mice,
WD-fed Klrk1⫺/⫺ApoE⫺/⫺ mice exhibited significantly
lower levels of serum alanine aminotransferase activities
(Figure 5A), indicating that their liver dysfunction was
alleviated. Furthermore, liver explants from Klrk1⫺/⫺ApoE⫺/⫺
mice, cultured ex vivo, produced significantly less IL-6 than
those from ApoE⫺/⫺ mice, indicative of reduced inflammation (Figure 5B). In addition, the numbers of macrophages,
NKT cells, and NK cells were also significantly lower in
livers of Klrk1⫺/⫺ApoE⫺/⫺ mice than ApoE⫺/⫺ mice (Figure
5C). These data suggest that NKG2D-mediated immune
activation is directly involved in liver inflammation and
dysfunction, which is known to promote abnormal metabolic
conditions.
NKG2D/Ligand Interaction Implicates Multiple
Immune Cell Populations in Liver Inflammation
To gain insight into cellular mechanisms that underlie
NKG2D-mediated liver inflammation, we determined the
influence of NKG2D deficiency on activation of different
immune cell populations in the WD-fed ApoE⫺/⫺ mice. The
cell types most likely to be affected are those that express
NKG2D, including NK and NKT cells in the liver (Figure IV
in the online-only Data Supplement). Indeed, NKT cells and,
to a lesser extent, NK cells from livers of the ApoE⫺/⫺ mice
exhibited considerable production of IFN-␥, which was
markedly attenuated when the mice lacked NKG2D (Figure
6A and 6B). In contrast, few conventional ␣␤ T cells of the
liver expressed IFN-␥ (not shown), and the liver macrophages
produced high levels of IL-6 in ApoE⫺/⫺ mice whether or not
the mice expressed NKG2D (Figure 6C). These results
suggest that the production of IL-6 by macrophages is
independent of NKG2D, consistent with the fact that they did
not express NKG2D (Figure IV in the online-only Data
Supplement). However, because liver macrophages were
markedly reduced in Klrk1⫺/⫺ApoE⫺/⫺ mice and they expressed NKG2D ligands in atherogenic conditions (Figures
2E and 2F and 5C), these cells likely stimulate NKG2Dmediated liver inflammation and at the same time are influenced by it.
Figure 5. Preventing NKG2D/ligand interactions alleviates
abnormal metabolic conditions by reducing liver inflammation in
the Western diet–fed apolipoprotein E– deficient (ApoE⫺/⫺)
model of atherosclerosis. A, Reduced serum alanine aminotransferase (ALT) activity in Western diet–fed Klrk1⫺/⫺ApoE⫺/⫺
mice. The experiment included 5 mice per group and was representative of 2 repeated experiments of a total of 10 mice. B,
Reduced interleukin (IL)-6 production by ex vivo cultured liver
explants of Klrk1⫺/⫺ApoE⫺/⫺ mice. Experiment includes 3 mice
per assay and was representative of 2 repeated experiments of a
total of 6 mice. C, Reduced numbers of immune cells in livers of
Klrk1⫺/⫺ApoE⫺/⫺ mice. The numbers are of each type of immune
cells per liver, calculated on the basis of total mononucleocytes
isolated from livers and percentages of each type. The experiment
included 3 mice per assay and was representative of 3 repeated
experiments of a total of 9 mice. WT indicates wild-type.
NKG2D in aggravating metabolic conditions may also contribute to the atherosclerosis.
Because the liver is the major organ of lipid metabolism
and exhibited highly upregulated expression of NKG2D
Discussion
The studies presented here identify the NKG2D/ligand axis as
a molecular link between chronic inflammation and metabolic
dysfunction that is critical for atherosclerosis and contributes
to type 2 diabetes mellitus progression. When it is considered
that the NKG2D ligands were upregulated in multiple tissues,
it can be seen that NKG2D-mediated immune activation
likely promotes atherosclerosis through several mechanisms.
In aortas, preferential upregulation of the NKG2D ligands is
associated with enhanced plaque formation in arch regions,
suggesting that it is directly involved in atherosclerotic
progression. NKG2D/ligand-mediated immune activation
could enhance the inflammation in atherosclerotic aortas,
which in turn might affect recruitment, survival, and function
of ligand-expressing macrophages that are directly involved
in plaque progression.40 Although upregulation of NKG2D
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Xia et al
NKG2D Promotes Atherosclerosis
2941
Figure 6. Multiple immune cell types are
involved in the liver inflammation mediated by NKG2D interactions. A, Comparison of the production of interferon
(IFN)-␥ by liver NKT cells of Western
diet–fed Klrk1⫺/⫺apolipoprotein E– deficient (ApoE⫺/⫺) and ApoE⫺/⫺ mice. Cells
are gated on TCR␤⫹NK1.1⫹ cells. B,
Comparison of IFN-␥ production by liver
natural killer (NK) cells of Western diet–
fed Klrk1⫺/⫺ApoE⫺/⫺ and ApoE⫺/⫺ mice.
Cells are gated on CD3⫺NK1.1⫹ cells. C,
On a per cell basis, NKG2D deficiency
did not affect interleukin (IL)-6 production
by liver CD11b⫹F4/80⫹ macrophages of
Western diet–fed ApoE⫺/⫺ mice. The
experiment in A through C included 3
mice per group and is representative of
3 repeated experiments of a total of 9
mice. The number below the gate in
each graph is the mean⫾SE percentage
of the positive cells of total cells in the
plot. The isotype-control staining was
similar in wild-type (WT), ApoE⫺/⫺, and
Klrk1⫺/⫺ApoE⫺/⫺ mice. ***, *Statistical
significance compared with values from
ApoE⫺/⫺ mice.
ligands is most profound in developed plaques, we found
that enhanced expression of the ligands was apparent in
aortas of ApoE⫺/⫺ mice as early as 8 weeks of age, before
plaques were detected (data not shown), suggesting that the
NKG2D/ligand axis might be involved in early stages of
atherosclerosis.
NKG2D/ligand-mediated immune activation could also
promote atherosclerosis by exacerbating metabolic dysfunctions and causing systemic inflammation in the liver and
potentially other tissues. In this regard, the abnormal upregulation of NKG2D ligands in the pancreas of mice with
nonobese diabetes, an autoimmune type 1 diabetes mellitus
model, reportedly contributes to immune destruction of
␤-cells and development of diabetes mellitus.24 Although the
underlying mechanisms are different, NKG2D also plays a
role in progression of type 2 diabetes mellitus in the studies
presented here. Therefore, the NKG2D/ligand axis is extensively involved in multiple aspects of diabetes mellitus and
atherosclerosis. It will be interesting to determine whether the
NKG2D/ligand axis is involved in the development of other
diabetes mellitus–associated complications, such as retinopathy, nephropathy, and neuropathy.
It is likely that NKG2D exerts its disease-causing effects in
multiple cell types. We observed a marked reduction in NK
and NKT cell activation in livers of WD-fed ApoE⫺/⫺ mice,
suggesting that NKG2D/ligand interactions may be necessary
for activating these cells and that such activation may
promote metabolic dysfunctions and atherosclerosis. Because
of the very limited numbers of cells, we could not perform a
similar analysis of cells isolated from atherosclerotic plaques.
Both NK and NKT cells have been reported to contribute to
the severity of atherosclerosis in similar mouse models.
Although it is difficult to compare different studies directly,
the published studies suggest that the contribution of NK or
NKT cells individually to atherosclerosis is less marked than
the contribution of NKG2D shown here. Therefore, it is
possible that both populations, along with other NKG2D⫹
cells such as ␥␦T cells, contribute to the disease in an
NKG2D-dependent fashion and that NKG2D deficiency
therefore has a greater effect on the disease than either of the
NKG2D⫹ cell populations do separately.
We also found that the NKG2D ligands are upregulated in
blood and plaques of patients with type 2 diabetes mellitus,
suggesting the potential clinical application of blocking
NKG2D/ligands in treatment of atherosclerosis or metabolic
disease in patients. However, additional preclinical studies
are necessary before this approach can be considered for
clinical use. It remains to be determined whether blockade of
NKG2D affects established plaques, for example. Another
issue that needs further study is whether binding of NKG2D
antibody to NKG2D induces global inhibition of the functions of NKG2D-expressing cells, which was suggested by
some studies41 but refuted by others.42 Although we cannot
rule out that this occurs in our antibody blockade studies, the
NKG2D knockout experiments provide a direct and compelling line of evidence that NKG2D is involved in the disease.
In conclusion, our findings provide new insights into the
mechanisms underlying association of inflammation with
atherosclerosis and type 2 diabetes mellitus and suggest
possible new approaches to therapy of these diseases. In
addition, the observation that sMICA is elevated in sera of
diabetic patients suggests that sMICA measurement may
have utility as a biomarker of atherosclerotic progression.
Acknowledgments
Dr Xia performed all of the experiments except the immunohistochemical staining. Drs Shi and Sukhova performed the immunohistochemical staining of human and mouse tissues. Dr Yang was
involved in the immunohistochemical staining of mouse tissues. Dr
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2942
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December 20/27, 2011
Miller was involved in analysis of blood of patients with diabetes
mellitus. Drs Raulet and Guerra generated NKG2D antibodies and
NKG2D knockout mice. Dr Xiong initiated the project, and Drs
Xiong, Xia, and Raulet designed the experiments, analyzed data, and
prepared the manuscript.
Sources of Funding
This study was supported by an institutional fund of Pennsylvania
State University, a fund from the Penn State Institute of Diabetes and
Obesity (to Dr Xiong), and grants from the National Institutes of
Health (to Dr Raulet).
Disclosures
Dr Raulet received grant support from Novo Nordisk. Drs Xiong, Xia,
and Raulet received payments from Novo Nordisk through Pennsylvania State University and University of California, Berkeley. Drs Guerra,
Yang, Shi, Sukhova, and Miller report no conflicts.
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CLINICAL PERSPECTIVE
It is well established that immune activation is involved in progression of atherosclerosis in patients with diabetes mellitus.
However, molecular events stimulating the immune activation under diabetic conditions are not well understood, hindering
our effort to modulate the immune system for prevention and/or treatment of the disease. We report herein that a group
of stimulating ligands for the immune-activating receptor NKG2D were upregulated in tissues such as aorta and liver in
diabetic mice or patients and that inhibiting the NKG2D/ligand interaction suppressed atherosclerosis and metabolic
dysfunction by preventing NKG2D-expressing immune cell–mediated tissue inflammation in model mice. These findings
suggest the potential application of targeting NKG2D/ligands in prevention/treatment of atherosclerosis and abnormal
metabolic conditions in diabetic patients. Because expression of the NKG2D ligands could be upregulated in other affected
tissues, NKG2D/ligands might be also involved in other diabetes mellitus–associated complications, such as retinopathy
and nephropathy. Furthermore, our finding that preventing the NKG2D/ligand-mediated inflammation could suppress
atherosclerosis even under constantly severe abnormal metabolic conditions suggests that immune activation contributes
to atherosclerosis through separate, although overlapping, processes than metabolic dysfunction does. Therefore, combined
treatment of both metabolic dysfunction and immune activation might represent a better therapeutic strategy than the
treatment of either symptom alone. Finally, our observation that levels of soluble NKG2D ligands were elevated
differentially in blood of patients with type 2 diabetic mellitus suggests its potential utility as a biomarker in predicting
progression of atherosclerosis and other complications.
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SUPPLEMENTARY MATARIAL
Supplementary Table 1. Clinical characteristics of sMICA-positive vs. sMICA-negative type 2
diabetic patients.
ID#
MICA+
patients
P values
sMICA
(pg/ml)
Age
(Yrs)
Years
with
diabetes
1
2
12250
2100
41
66
1
10
158
159
3
4
598
533
59
59
4
9
264
157
5
6
7
440
431
350
62
63
47
1
1
1.5
124
279
94
8
9
156
91
67
60
4
1
134
158
10
‡
Avg
±SD
39
1698
±3753
68
59.2
±8.7
0.63
3
3.6
±3.4
0.29
131
165.8
±59.5
0.61
57.3
±9.2
58
61
62
6.1
±5.3
4
2
3
157.2
±47.2
101
206
132
56
41
2
5
151
201
68
2
113
62
10
125
60
2
154
‡
Avg
±SD
11
12
13
14
15
MICApatients
†
BD
BD
BD
BD
BD
BD
BD
16
Blood
glucose
(mg/dL)
BD
17
BD
18
BD
19
20
21
BD
BD
41
68
63
10
20
5
129
118
205
22
BD
48
4
251
Medical
Condition
high BP*
heart disease
High BP,
Osteoporosis
gall bladder
Kidney Stones
none noted
high cholesterol
high BP, high
cholesterol
none noted
high cholesterol,
high BP
high cholesterol
high BP
Stents, high BP
high cholesterol
Heart palpatations
high cholesterol,
high BP
high cholesterol,
high BP
high BP, high
cholesterol
high BP, high
cholesterol
high cholesterol
high BP
high BP, high
cholesterol
Medication
Zocor (simvastatin)
none noted
Zocor (simvastatin)
Zocor (simvastatin)
Vytorin
(simvastatin,
ezetimibe)
none noted
none noted
Zetia (ezetimibe)
simvastatin
Lovastat
Zetia, Avandia
none noted
Zetia (ezetimibe)
Lipitor
(atorvastatin)
none noted
none noted
Lipitor
(atorvastatin)
none noted
none noted
Zocor (simvastatin)
none noted
Zocor (simvastatin)
Note: Ages of the healthy controls (N=19) are 52.1±2.5 years and no medications are noted for
them.
*high BP: high blood pressure;
†BD: below the detection limit;
‡
Avg±SD: Average± standard deviation.
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Supplementary Figure 1. Genotyping of Klrk1-/-ApoE-/- mice. For genotyping of ApoE
knockouts, genomic DNA was PCR amplified with a set of three primers that produced a 245-bp
band of the ApoE knockout allele and a 155-bp band of the wild type ApoE allele in one PCR
reaction. Sequences of the three primers used for the ApoE knockout genotyping were
0IMR0180:GCC TAG CCG AGG GAG AGC CG; 0IMR0181: TGT GAC TTG GGA GCT
CTG CAG C; and 0IMR0182: GCC GCC CCG ACT GCA TCT (The Jackson laboratory). For
genotyping of Klrk1 knockouts, genomic DNA was PCR amplified with a set of three primers
that yielded a 422-bp band for the wild-type Klrk1 allele and a 317-bp band for the Klrk1
knockout allele in one PCR reaction. The three primers used for the Klrk1 genotyping were
published28. WT: wild type. KO: knockout.
2
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Supplementary Figure 2. Real-time RT-PCR analysis of transcripts for the NKG2D ligands
Rae-1δ, Rae-1ε and H60b in arch and thoracic regions of aortae of same 8 month-old ApoE-/mice feeding on normal chow. The results are presented as ratios of the expression levels in the
arch vs. thoracic regions of aortae. The expression levels of the NKG2D ligands are normalized
on the β-actin levels. **P<0.01 and ***P<0.001 are calculated based on comparison of the
expression levels of the individual NKG2D ligands in the arch vs. thoracic regions of aortae,
averaged of three mice.
3
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Supplementary Figure 3. Real-time RT-PCR analysis for transcripts of different NKG2D
ligands in wild-type macrophages cultured in vitro in presence of oxLDL or nLDL. The culture
condition of the macrophages was described in the Method section. The results are of three
assays. **P<0.01, ***P<0.001.
4
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Supplementary Figure 4. The NKG2D expression on various subsets of immune cells isolated
from livers, spleens and aortae of WD-fed ApoE-/- or normal wild type mice. Mononucleocytes
isolated from livers or aortae or splenocytes were immunofluorescently stained and analyzed by
flow cytometry for NKG2D expression on gated macrophages (CD11b+F4/80+), NKT
(CD3+NK1.1+), NK (CD3-NK1.1+) and conventional αβT cells (CD3+NK1.1-TCRβ+). The gray
areas are of staining with isotype control antibodies. In the samples of livers, the cells of
NKG2D-knockout Klrk1-/-ApoE-/- mice are included as additional negative controls for the
NKG2D staining. The experiment was done twice. Few NK and NKT cells could be isolated
from aortae of normal wild type mice for the reliable analysis and not shown.
5
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Supplementary Figure 5. Oil red O staining to detect lipid-deposition in aortic roots of WD-fed
(A) or STZ-induced diabetic (B) ApoE-/- and Klrk1-/-ApoE-/- mice. Plaque areas stained red.
Representatives of the staining of each type were shown in the left and middle panels.
Quantitative comparison of Oil red O staining positive areas of each type was shown in the right
panels, compiled from results of four to five mice for each type.
6
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Supplementary Figure 6. Clearer appearance of sera of WD-fed Klrk1-/-ApoE-/- mice than of
similarly fed ApoE-/- mice.
7
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