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Regulatory Role of Dendritic Cells in Post-Infarction Healing and Left

Ventricular Remodeling

Running title: Anzai et al.; Dendritic cells in LV remodeling after MI

Atsushi Anzai, MD1; Toshihisa Anzai, MD, PhD1,4; Shigenori Nagai, PhD2,5;

Yuichiro Maekawa, MD, PhD1; Kotaro Naito, MD, PhD1; Hidehiro Kaneko, MD1;

Yasuo Sugano, MD, PhD1,6; Toshiyuki Takahashi, MD, PhD1; Hitoshi Abe, BSc3;

Satsuki Mochizuki, PhD3; Motoaki Sano, MD, PhD1; Tsutomu Yoshikawa, MD, PhD1;

Yasunori Okada, MD, PhD3; Shigeo Koyasu, PhD2; Satoshi Ogawa, MD, PhD1,6;

Keiichi Fukuda, MD, PhD1

1Division of Cardiology, Dept of Med, 2Dept of Microbiology & Immunology, 3Dept of Pathology, Keio University School of Med, Tokyo, Japan; 4Dept of Cardiovascular Med,

National Cerebral & Cardiovascular Center, Osaka, Japan; 5Core Research for Evolutional Science & Technology (CREST), Japan Science & Technology Agency (JST); 6Cardiovascular

Center, International University of Health & Welfare Mita Hospital, Tokyo, Japan

Correspondence: Toshihisa Anzai, MD, PhD Department of Cardiovascular Medicine National Cerebral and Cardiovascular Center 5-7-1 Fujishiro-dai, Suita Osaka 565-8565, Japan Phone: +81-6-6833-5012 (ext.2321) Fax: +81-6-6833-9865 E-mail: [email protected]

Journal Subject Codes: [148] Heart failure - basic studies

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Abstract:

Background- Inflammation and immune responses are integral components in the healing

process after myocardial infarction (MI). We previously reported dendritic cell (DC) infiltration

in the infarcted heart. However, the precise contribution of DC in post-infarction healing is

unclear.

Methods and Results- Bone-marrow (BM) cells from CD11c-diphtheria toxin receptor/GFP

transgenic mice were transplanted into lethally irradiated wild-type recipient mice. After

reconstitution of BM-derived cells, the recipient mice were treated with either diphtheria toxin

(DC-ablation) or vehicle (control), and MI was created by left coronary ligation. CD11c+ GFP+

DCs expressing CD11b and MHC class II were recruited into the heart, peaking on day 7 after

MI in control group. Mice with DC-ablation for 7 days showed deteriorated left ventricular

function and remodeling. DC-ablated group demonstrated enhanced and sustained expression of

inflammatory cytokines such as interleukin (IL)-1 , IL-18 and tumor necrosis factor- ,

prolonged extracellular matrix degradation associated with a high level of matrix

metalloproteinase-9 activity, and diminished expression level of IL-10 and endothelial cell

proliferation following MI compared with control group. In vivo analyses revealed that

DC-ablated infarcts had enhanced monocyte/macrophage recruitment. Among these cells,

marked infiltration of proinflammatory Ly6Chigh monocytes and F4/80+ CD206- M1

macrophages, and conversely impaired recruitment of anti-inflammatory Ly6Clow monocytes and

F4/80+ CD206+ M2 macrophages in the infarcted myocardium were identified in DC-ablated

group than in control group.

Conclusions- These our results suggest that dendritic cell is a potent immunoprotective regulator

during the post-infarction healing process, via controlling monocyte/macrophage homeostasis.

Key words: myocardial infarction, remodelling, inflammation, immune system, heart failure



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Introduction

Left ventricular (LV) remodeling after myocardial infarction (MI) is the process of

complex architectural myocardial alteration and is associated with a poor clinical outcome.

Congestive heart failure due to post-infarction LV remodeling remains an unresolved problem in

spite of aggressive revascularization and pharmacological therapy in recent clinical practice.

Therefore, better understanding of the molecular and cellular mechanisms involved in this

process and the search for alternative therapeutic targets against LV remodeling are matters of

great importance.

Inflammation and immune responses are integral components of the host reaction to

myocardial injury, and play a crucial role in infarct healing and subsequent LV remodeling.1-3 We

have previously reported that elevated serum C-reactive protein concentration and peripheral

monocytosis predict a poor clinical outcome after MI.4, 5 Moreover, our experimental study

demonstrated that the enhanced infiltration of monocytes and macrophages into the infarcted

myocardium induced by granulocyte-macrophage colony-stimulating factor leads to aggravated

infarct expansion and LV dysfunction.6 These findings indicate that excessive immune-mediated

inflammatory reactions have a deleterious effect on post-infarction LV remodeling. On the other

hand, immunosuppressive therapy using corticosteroids resulted in increased catastrophic

incidents such as cardiac rupture in clinical practice,7, 8 and recent experimental data showed that

macrophage depletion with clodronate-containing liposomes (Clo-Lip) impaired infarct healing

in a murine model,9 suggesting that controlled inflammation and immune response are

prerequisite for appropriate cardiac repair after MI. However, the regulatory mechanism

controlling these reactions during the post-infarction healing process remains to be determined.

The dendritic cell (DC) is a potent central immunoregulator, which orchestrates various



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kinds of inflammatory cells in innate and adoptive immunity.10-12 After microbial infection or

tissue injury, bone-marrow (BM) and splenic precursors and circulating monocytes are reported

to differentiate into DCs and exert various influences on the immune system at the inflammatory

site, such as priming of antigen-specific immune responses, induction of tolerance, and chronic

inflammation.13-15 Zhang et al. first demonstrated infiltration of DCs into the infarcted heart in

experimental MI.16 We have previously reported that an increased number of mature DCs in the

infarcted heart was associated with deterioration of LV remodeling in a rat MI model.17 However,

the causative effect of infiltrating DCs on LV remodeling and their origin in the post-infarction

healing process are unclear.

The primary aim of this study was to clarify the role of DCs in tissue repair and LV

remodeling after MI and the origin of DCs involved in the process. We employed transgenic

mice expressing diphtheria toxin receptor (DTR) on DCs, enabling us to specifically deplete DCs

by injecting diphtheria toxin (DT), which have already been proven to be a powerful and useful

tool to manipulate DCs in vivo.18-20

Methods

An additional detailed Methods section can be found in the online-only Data

Supplement.

Animals

CD11c-DTR/GFP transgenic mice on a C57BL/6 background (CD45.2+) were provided

by D. Littman (New York University, NY). B6.SJL congenic mice on a C57BL/6 background

(CD45.1+) were obtained from Taconic (Germantown, NY). Wild-type (WT) C57BL/6 mice

(CD45.2+) were purchased from Clea Japan (Tokyo, Japan). These mice were bred and kept



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under specific-pathogen-free conditions. All experiments were performed in accordance with the

Keio University animal care guidelines, which conform to the Guide for the Care and Use of

Laboratory Animals published by the US National Institutes of Health (NIH Publication No.

85-23, revised 1996).

Bone-marrow (BM) Cell Preparation and BM Transplantation (BMT)

A BMT model was used to evaluate the role of DCs by depleting CD11c+ DTR+ cells by

DT administration.19 The detailed methods to harvest BM cells and to perform BMT are

described in the online-only Data Supplement.

DT Administration

BMT mice were intraperitoneally inoculated with DT (Sigma, St. Louis, MO) in

phosphate buffered saline (PBS) at a dose of 4 ng/g body weight. BMT mice were randomly

assigned to DT (DC-ablation) and PBS (control) treatment groups. DT or PBS was administered

on the day before MI or sham operation and then 2 and 5 days after the operation. In this study,

control and DC-ablated BMT mice were compared in permanent coronary occlusion model

according to the methods section mostly described in online-only data supplement.

Additional Methods

The expanded Methods section in the online-only Data Supplement contains

information on BM cell preparation and BMT; creation of MI; echocardiography and

hemodynamics; morphometric analysis; real-time quantitative polymerase chain reaction;

Western blotting; gelatin zymography; immunohistochemical staining; immunofluorescent

staining; preparation of splenic and peripheral blood cells; isolation of infiltrating leukocytes

from the infarcted heart; and flow cytometry.

Statistical Analyses



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All data were expressed as mean value SEM. Comparisons between two groups were

performed using Wilcoxon rank sum test. When more than two groups were analyzed,

Kruskal-Wallis test followed by Bonferroni’s post-hoc test was employed. Survival distributions

were estimated using the Kaplan-Meier method and compared by the log-rank test. Difference in

cause of death between control and DC-ablated mice after MI was assessed by Fisher’s exact test.

A P value of < 0.05 was considered to be significant. All statistical analyses were performed with

SPSS 15.0 statistical software package for Windows (SPSS Inc., Chicago, IL).

Results

Strategy of DC Depletion In Vivo

To minimize the effect of DT on non-hematopoietic cells expressing an integrin, CD11c,

we employed a BMT model as described in Methods. The repopulation efficiency of

hematopoietic cells after BMT was determined using B6.SJL mice (CD45.1+) as recipients. After

reconstitution with donor CD11c-DTR/GFP transgenic marrow cells (CD45.2+), flow cytometric

analysis of splenocytes revealed that the reconstitution rate of our BMT method was

approximately 96% (Supplemental Figure 1A). Moreover, almost all the CD11c+ DCs were

CD45.2+ (CD45.2- CD11c+ recipient-derived DCs were absent) and more than 97% of these DCs

were also GFP+ (Supplemental Figure 1B). These data indicated that cells such as DCs of BMT

mice were almost totally replaced with donor BM-derived cells in our BMT mice.

We assessed the effect of DT inoculation in BMT mice by flow cytometry. Flow

cytometric analysis of splenocytes revealed that CD11c+ GFP+ and CD11c+ MHC class II

(MHCII)+ DCs were completely depleted by DT administration (Supplemental Figure 2A),

whereas other immune cells, CD4+ T cells, CD8+ T cells, CD19+ B cells, and CD11b+



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monocytes/macrophages, were hardly affected (Supplemental Figure 2B). These data were

compatible with other previously published papers using this transgenic mouse system.18, 19

Recruitment of DCs into the Infarcted Heart

Peripheral circulating CD11c+ GFP+ DCs expressing MHCII rapidly increased after MI

in control mice, and were almost completely abolished by DT administration in DC-ablated mice.

Immunofluorescent staining showed that resident DCs were rarely observed in the control heart

without MI and the number of heart-infiltrating CD11c+ GFP+ DCs in the infarcted myocardium

reached its peak 7 days after MI in control mice, but they were absent in DC-ablated mice for 7

days after MI (Figure 1A through 1C). DCs were predominantly identified in the infarcted and

border area, whereas there were few DCs in the non-infarcted area after MI in control mice.

These DCs infiltrating the heart also expressed CD11b and MHCII, which almost completely

disappeared in DC-ablated mice 7 days after MI (Figure 1D). These data indicate that infiltrating

cardiac mature DCs originated from the BM and were recruited into the infarcted heart, and that

DT treatment is effective for the abrogation of DC infiltration after MI in our BMT model.

No effects of DT on WT mice were confirmed by the data of echocardiographic and

organ weight measurements 28 days after sham or MI operation, showing that LV function and

heart/lung weight adjusted for body weight were almost similar in non-treated and DT-treated

WT mice (Supplemental Figure 3).

DC-Ablation Aggravates LV Dysfunction and Remodeling after MI

Cardiac function was analyzed 28 days after MI (Table 1). Echocardiographic

measurements revealed acceleration of cardiac dilatation and deterioration of LV function in

DC-ablated mice compared with control mice. Hemodynamic data also indicated that maximum

rate of isovolumic pressure development after MI was significantly lower in DC-ablated mice



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than in control mice. Moreover, heart and lung weights adjusted for body weight 28 days after

MI were significantly higher in DC-ablated mice compared with control mice.

Acceleration of MI-Induced Death, Wall Thinning and Myocardial Fibrosis in DC-Ablated

Mice

The survival rate tended to be lower in DC-ablated mice than in control mice 28 days

after MI, whereas all sham-operated animals in both groups survived throughout the study

(Figure 2A). We found that the incidence of death from heart failure tended to be higher in

DC-ablated mice than in control mice after MI (21.7% vs. 9.4%, P = 0.086), although the

occurrence of cardiac rupture was similar in the two groups (13.0% vs. 9.4%, P = 0.53).

Masson’s trichrome staining revealed that there was no difference in the gross morphology in

sham-operated hearts between control and DC-ablated mice (Figure 2B). Twenty-eight days

after MI, DC-ablated mice demonstrated enhanced infarct expansion and wall thinning compared

with control mice, although the infarct size was similar in the two groups (Figure 2B through

2D). Collagen volume fraction detected by picrosirius red staining in the non-infarcted area was

significantly higher in DC-ablated mice compared with control mice 28 days after MI (Figure

2E and 2F). Myocardial fibrosis in the infarcted area did not significantly differ between

DC-ablated and control mice 28 days after MI (Figure 2E and 2F). However, picrosirius red

polarized microscopy of collagen fibers in the infarcted area showed that a predominance of

loosely assembled green or yellow fibers were seen in DC-ablated mice, whereas the well

aligned thick orange fibers were observed in control mice 28 days after MI (Figure 2G).

Absence of DCs Enhances Inflammatory Cytokine but Suppresses Anti-Inflammatory

Cytokine Expression after MI

Figure 3 demonstrates the temporal changes of interleukin (IL)-1 , IL-18, tumor



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necrosis factor (TNF)- , IL-10, CCL2 and CX3CL1 expression after MI. In control mice, the

expression of IL-1 , IL-18, TNF- , and CCL2, which are proinflammatory, increased 3 days

after MI and gradually decreased thereafter (Figure 3A through 3D). In contrast, DC-ablated

mice had sustained elevation of IL-1 , IL-18, TNF- , and CCL2 for 7 days after MI compared

with control mice (Figure 3A through 3D). On the other hand, the expression of IL-10 and

CX3CL1, which are anti-inflammatory, 7 days after MI was lower in DC-ablated mice than in

control mice (Figure 3E and 3F). Western blotting analysis confirmed that expression of IL-10

at the protein level was significantly lower in DC-ablated mice compared with control mice 7

days after MI (Figure 3G and 3H).

DC-Ablation Accentuates Matrix Metalloproteinase (MMP)-9 Activity and Inducible Nitric

Oxide Synthase (iNOS) Expression after MI

As shown in Figure 4, MMP-9 activity was increased in the infarcted heart 3 days after

MI and decreased thereafter in control mice, whereas its activity in DC-ablated mice was

persistently upregulated for 28 days after MI (Figure 4A and 4B). The activity of MMP-2 tended

to be higher in DC-ablated mice than in control mice, although there was no statistical

significance between the two groups (Figure 4A and 4C). Furthermore, iNOS was also increased

by MI, and DC-ablation further enhanced the induction of iNOS 7 days after MI (Figure 4D and

4E).

Post-Infarction Myocardial Angiogenesis is Impaired by DC Depletion

To assess the influence of DC depletion on cardiac angiogenesis, we performed the

immunoblot and histological analyses. As shown in Figure 5A and 5B, the expression of

vascular endothelial growth factor (VEGF) at protein level, which was upregulated in control

infarcts, was significantly suppressed by DC-ablation 7 days after MI. Moreover, our



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immunohistochemical staining revealed that the number of blood vessels in the infarcted

myocardium was smaller and the fraction of CD31+ Ki67+ proliferating endothelial cells was

significantly lower in DC-ablated mice compared with control mice 7 days following MI (Figure

5C and 5D), suggesting that DC depletion might affect the cardiac neoangiogenesis during the

post-infarction healing process.

Enhanced Infiltration of Inflammatory M1 Macrophages and Diminished Recruitment of

Anti-Inflammatory M2 Macrophages into the Infarcted Myocardium in Response to

DC-Ablation

We evaluated the degree of various inflammatory cell infiltration after MI in control and

DC-ablated mice. The number of total CD45+ leukocytes in the infarcted heart was significantly

higher in DC-ablated mice than in control mice 7 days after MI (Supplemental Figure 4A).

Immunofluorescent staining also indicated that infiltration of CD45+ leukocytes into the

infarcted myocardium 7 days after MI was greater in DC-ablated mice compared with control

mice (Supplemental Figure 4B). Immunohistochemical staining for Mac3 showed that the

number of infiltrating differentiated macrophages into the infarcted myocardium peaked on day 7

after MI and was significantly higher in DC-ablated mice 7 and 14 days after MI compared with

control mice (Figure 6A and 6B). Flow cytometric analysis also demonstrated that the

percentage of Mac3+ macrophages in the heart 7 days after MI in DC-ablated mice was

significantly higher compared with control mice (Supplemental Figure 4C and 4D).

Furthermore, among tissue-infiltrating F4/80+ macrophages, DC-ablated infarcts had enhanced

infiltration of F4/80+ CD206- inflammatory M1 macrophages and diminished recruitment of

F4/80+ CD206+ anti-inflammatory M2 macrophages compared with control mice 7 days after MI

based on the flow cytometry (Figure 6C and 6D). The number of MPO+ neutrophils in the



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infarcted heart 7 days after MI tended to be higher in DC-ablated mice compared with control

mice (Figure 6E). However, the number of CD3+ T cells infiltrating the infarcted myocardium

was comparable in the two groups (Figure 6F).

DC-Ablation Causes Increased Ly6Chigh Monocyte but Decreased Ly6Clow Monocyte

Infiltration into the Infarcted Heart

Monocytes, which play important roles in tissue repair after MI, were evaluated in

peripheral blood and heart tissue. Flow cytometric analysis showed that the total number of

peripheral blood monocytes was higher in DC-ablated mice compared with control mice

(Supplemental Figure 5A). In addition, peripheral blood Ly6Chigh monocytes were persistently

increased while Ly6Clow monocytes were decreased in DC-ablated mice compared with control

mice, especially 7 days after MI (Figure 7A and 7B). Moreover, among the inflammatory

leukocytes isolated from the infarcted heart, the number of total monocytes and inflammatory

Ly6Chigh monocytes were markedly higher whereas the number of reparative Ly6Clow monocytes

was lower in DC-ablated mice 7 days after MI compared with those in control mice

(Supplemental Figure 5B, Figure 7C and 7D).

Discussion

We demonstrated here that mature, activated CD11c+ CD11b+ DCs originating from the

BM infiltrate the infarcted heart during the healing process after MI. Selective depletion of DCs

exacerbated post-infarction LV remodeling in association with enhanced inflammatory cytokine

expression, iNOS production, and MMP-9 activation likely via marked infiltration of

proinflammatory Ly6Chigh monocytes and F4/80+ CD206- M1 macrophages into the infarcted

myocardium. Meanwhile, the number of anti-inflammatory Ly6Clow monocytes and F4/80+



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CD206+ M2 macrophages, myocardial expression of IL-10 and cardiac angiogenesis after MI

were suppressed by selective ablation of DCs. These results indicated the protective role of DCs

in the post-infarction inflammation and subsequent LV remodeling by regulation of cellular

employment in the heart.

Leukocyte influx is a highly sophisticated system contributing to cardiac repair

following MI. Inflammatory signals recruit neutrophils to the ischemic heart immediately after

MI, and monocytes/macrophages shortly thereafter. A large number of reports have indicated

that excessive cardiac macrophage infiltration is associated with facilitated infarct expansion and

adverse LV remodeling with augmented inflammation and MMP activity.6, 21, 22 Macrophages

can be divided into two populations, which have a classically (M1) and alternatively (M2)

activated phenotype.23 In addition to macrophages, growing evidence has emerged that

monocytes, classically recognized as the precursor of tissue-infiltrating differentiated

macrophages, per se have an important role in the post-infarction healing process. In the mouse,

there are two distinctive subsets of monocytes; CD11bhigh Ly6Chigh proinflammatory monocytes

and CD11bhigh Ly6Clow reparative monocytes.24, 25 Nahrendorf et al. reported that the

post-infarction repair process consists of biphasic reactions, in which the early phase is

dominated by Ly6Chigh monocytes that are recruited in a CCL2-CCR2-dependent manner and

exhibit phagocytic, proteolytic, and inflammatory properties, whereas the late phase is dominated

by Ly6Clow monocytes that are recruited in a CX3CL1-CX3CR1-dependent manner and promote

angiogenesis and resolution of inflammation.26 Moreover, they also demonstrated that Ly6Chigh

monocytosis observed in apolipoprotein E knockout atherosclerotic mice was associated with

exacerbated post-infarction LV remodeling through increased inflammatory reaction and MMP

activity, whereas in vivo depletion of neutrophils did not improve LV dysfunction after MI.27 On



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the other hand, infiltrating macrophage depletion by intravenous injection of Clo-Lip markedly

impaired wound healing and accelerated LV remodeling and mortality after myocardial injury.9,

26 Furthermore, depletion of circulating Ly6Chigh monocytes by administration of Clo-Lip during

the early phase of MI caused larger areas of debris and necrotic tissue, whereas depletion of

Ly6Clow monocytes by late phase administration of Clo-Lip resulted in decreased collagen

deposition and reduced neoangiogenesis,9, 26 suggesting that successful cardiac repair depends on

well-coordinated recruitment of monocytes/macrophages in the injured heart. However, the

molecular or cellular mechanisms upstream of this critical framework remain to be elucidated.

As the conductor of immune-mediated inflammatory reactions, since their discovery in

1973, DCs have attracted a great deal of attention because of their distinctive and wide variety of

functions.28 DCs are of hematopoietic origin and are widely distributed in organs and tissues.

They are characterized by their capability of antigen capture, migration, antigen presentation and

activation of other immune cells, and control immunity and tolerance in various conditions, such

as infection, autoimmune disease, tumor, and allergy.10-12, 29 However, there are few reports

regarding the role of DCs in cardiovascular disease, especially in LV remodeling after MI.

Maekawa et al. demonstrated that activation of IL-1 receptor associated kinase (IRAK)-4, which

is a downstream molecule of IL-1 and toll-like receptor family members, in DCs adversely

affected post-infarction inflammation and LV remodeling, using IRAK-4-deficient mice.30

However, the IRAK-4 signaling pathway is also operative in cardiomyocytes and other immune

cells in addition to DCs.31, 32 Since the initial inflammatory signals after MI are known to involve

cardiomyocytes and are further strengthened by neutrophils and monocytes/macrophages

expressing IRAK-4, their results might reflect the involvement of these cells in the development

of post-infarction LV dysfunction. Therefore, the precise contribution of DCs in the infarcted



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heart remains unclear.

In the present study, we employed CD11c-DTR/GFP transgenic mice to examine the

effect of DC depletion on post-infarction myocardial remodeling. Conditional DC depletion for 7

days after MI resulted in dysregulation of organized monocyte/macrophage employment, critical

for the post-infarction repairing process. In vivo analyses revealed that the number of peripheral

monocytes and tissue monocytes/macrophages was higher in DC-ablated mice than in control

mice. Among these monocytes/macrophages, marked infiltration of inflammatory Ly6Chigh

monocytes and F4/80+ CD206- M1 macrophages and suppressed recruitment of

anti-inflammatory Ly6Clow monocytes and F4/80+ CD206+ M2 macrophages were identified in

DC-ablated mice, and this was associated with increased expression of CCL2 and decreased

expression of CX3CL1. Prolonged expression of inflammatory cytokines and iNOS and

enhanced MMP-9 activation were also observed in DC-ablated mice, which were associated with

increased peripheral blood and tissue inflammatory monocytes/macrophages. Consistent with

previously published paper which demonstrated that thick collagen fibers were degraded by

augmented MMPs and replaced by premature collagen fibers in the infarcted heart,33 our study

based on the picrosirius polarized microscopy showed that DC-ablated infarcts had disorganized

collagen fibers, which may lead to subsequent infarct expansion and heart failure. Moreover,

cardiac angiogenesis, which has a favorable effect on tissue repair following MI, was inhibited in

DC-ablated mice compared with control mice. As discussed above, Ly6Clow monocytes

selectively express higher levels of VEGF,26 and M2 macrophages also have proangiogenic

properties.34 Taken together, these findings indicate that the absence of DCs leads to activation

of inflammatory monocytes/macrophages and suppression of reparative monocytes/macrophages,

inducing enhanced inflammation, extracellular matrix degradation and apoptosis, and impaired



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neoangiogenesis in post-infarction healing process.

DCs, macrophages, and monocytes are members of the mononuclear phagocyte system,

defined as non-granulocytic, myeloid cells that play important roles in tissue remodeling and

homeostasis, as well as regulatory and stimulatory aspects of innate and adaptive immunity.35

Their origins are presumed to be the same BM precursor cells, named macrophage DC

progenitors.36, 37 Moreover, several reports have shown that peripheral monocytes transmigrate

into inflamed tissue and differentiate into macrophages or DCs.37-39 Based on such tight

interaction in the mononuclear phagocyte system, it is possible that selective DC depletion may

change the subpopulations of the system and that DCs contribute to the maintenance of

disciplined monocyte/macrophage organization following MI. Although the precise mechanisms

of the anti-inflammatory effect of DCs through controlling monocyte/macrophage subsets are not

clear, it may be very difficult to address the mechanism since immune cells communicate

through the release of many secreted factors. Further studies are needed to investigate how DCs

interact with monocytes/macrophages in the healing myocardium.

In summary, the present study demonstrated that DC is likely to play an

immunoprotective role especially in monocyte/macrophage homeostasis during the

post-infarction healing process, and suggests that modulation of DCs could be a new therapeutic

target in LV remodeling after MI.

Acknowledgements: We thank Hiromi Kato (Keio University), Mayu Matsuda (Keio

University), and Yukiko Baba (Keio University) for excellent technical assistance. Thanks are

also due to Kyoko Hidaka for animal care.

Funding Sources: This work was supported by a Grant-in-Aid for Young Scientists (B)

(23790875 to A.A. and 21790476 to S.N.), a Grant-in-Aid (20590872 to T.A.), and a Medical



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School Faculty and Alumni Grant from the Keio University Medical Science Fund (T.A.).

Conflict of Interest Disclosures: S.K. is a consultant for Medical and Biological Laboratories,

Co. Ltd.

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Table 1. Body and Organ Weights, Echocardiographic, and Hemodynamic Data 28 Days after MI Sham MI Control DC-ablation Control DC-ablation

Organ weight n 6 6 13 12 BW, g 27.2 0.3 26.8 0.4 24.6 0.3* 24.3 0.3* HW/BW, mg/g 3.66 0.06 3.63 0.05 4.35 0.13* 4.75 0.10*† LW/BW, mg/g 4.19 0.09 4.01 0.12 7.40 0.47* 8.90 0.47*† Echocardiography n 6 6 11 12 HR, bpm 535.9 4.8 532.0 4.8 524.1 6.0 521.1 3.1 AWT, mm 0.66 0.02 0.66 0.02 0.54 0.01* 0.52 0.02* PWT, mm 0.66 0.02 0.65 0.02 0.62 0.03 0.59 0.02 LVEDD, mm 2.74 0.06 2.79 0.06 4.22 0.12* 4.65 0.13*† LVESD, mm 1.36 0.03 1.39 0.03 3.11 0.10* 3.62 0.13*† FS, % 50.3 0.4 50.2 0.3 26.2 0.5* 22.3 0.8*† LV mass, mg 49.0 2.23 50.0 3.13 85.1 3.95* 95.9 4.21*† LV mass/BW 1.80 0.07 1.87 0.11 3.46 0.16* 3.94 0.16*† Hemodynamics n 6 6 13 12 HR, bpm 508.9 4.2 509.6 4.9 507.8 6.4 503.4 3.9 LVESP, mmHg 105.7 1.5 106.2 1.3 83.3 1.3* 77.6 1.3* +dP/dt, mmHg/s 11695 946 11881 847 7836 341* 6392 246*† -dP/dt, mmHg/s 9976 456 9740 367 5205±202* 4723±199* MI, myocardial infarction; DC, dendritic cell; BW, body weight; HW, heart weight; LW, lung weight; HR, heart rate; AWT, anterior wall thickness; PWT, posterior wall thickness; LVEDD, left ventricular end-diastolic diameter; LVESD, left ventricular end-systolic diameter; FS, fractional shortening; LVESP, left ventricular end-systolic pressure. Results are presented as mean ± SEM. *P < 0.05 vs corresponding sham group; †P < 0.05 vs control MI group.



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Figure Legends:

Figure 1. Recruitment of DCs into the infarcted heart. A, Representative photographs of

immunofluorescent staining for CD11c (red) and GFP (green) in the infarcted myocardium 7

days after MI in control and DC-ablated mice. Scale bars indicate 30 m. B, High magnification

view of rectangle in A. White arrows indicate CD11c+ GFP+ heart-infiltrating DCs (yellow).

Scale bar indicates 30 m. C, Time course of CD11c+ GFP+ DCs infiltration after MI in control

and DC-ablated mice analyzed by immunofluorescent staining. Data were obtained from 5

independent experiments at each time point. D, Representative flow cytometric dot plots of

heart-infiltrating CD45+ leukocytes for CD11c-APC and CD11b-PE or MHCII-PE in control and

DC-ablated mice 7 days after MI. *P < 0.05 and **P < 0.01 vs sham of same group; †P < 0.05 and ††P < 0.01 vs control MI.

Figure 2. DC-ablation accelerates MI-induced death, wall thinning and myocardial fibrosis

without affecting infarct size. A, Kaplan-Meier survival analysis in control and DC-ablated mice

after MI or sham operation. B, Representative Masson’s trichrome staining of cardiac tissue

sections in control and DC-ablated mice 28 days after MI or sham operation. Scale bars indicate

1 mm. C, Wall thickness of scar at papillary muscle level 28 days after MI in control (n = 11)

and DC-ablated (n = 12) mice. D, Quantitative analysis of infarct size 28 days after MI in control

(n = 11) and DC-ablated (n = 12) mice. E, Representative picrosirius red-stained sections of

infarcted and non-infarcted areas 28 days after MI in control and DC-ablated mice. Scale bars

indicate 100 m. F, Quantification of fibrotic area in infarcted and non-infarcted area 28 days

after MI in control (n = 11) and DC-ablated (n = 12) mice. G, Polarized microscopic view of

picrosirius red-stained section demonstrating in E. †P < 0.05 and ††P < 0.01 vs control MI.

Figure 3. Ablation of DC enhances proinflammatory cytokine but suppresses anti-inflammatory

cytokine expression in the infarcted myocardium. A through F, Time course of mRNA

expression of IL-1 (A), IL-18 (B), TNF- (C), CCL2 (D), IL-10 (E), and CX3CL1 (F) in the

infarcted heart after MI in control and DC-ablated mice. Data were obtained from 8 to 10

independent experiments at each time point. G, Representative immunoblotting image for IL-10

in the infarcted heart of control and DC-ablated mice 7 days after MI or sham operation. H,



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Quantitative analysis of IL-10 protein expression in control and DC-ablated mice 7 days after MI

or sham operation (n = 5 per group). *P < 0.05 and **P < 0.01 vs sham of same group; †P < 0.05

vs control MI.

Figure 4. Increased MMP-9 activity and iNOS expression in DC-ablated mice after MI. A,

Representative photograph of zymographic gel demonstrating time course of MMP-9 and -2

activities in the infarcted heart of control and DC-ablated mice. B and C, Quantitative analyses

of MMP-9 (B) and -2 (C) activities after MI based on gelatin zymography in control and

DC-ablated mice. Data were obtained from 5 independent experiments at each time point. D,

Representative immunoblotting analysis for iNOS in the infarcted heart of control and

DC-ablated mice 7 days after MI. E, Quantitative assessment for iNOS expression in the

infarcted heart of control and DC-ablated mice 7 days after MI (n = 5 per group). *P < 0.05 and **P < 0.01 vs sham of same group; †P < 0.05 and ††P < 0.01 vs control MI.

Figure 5. Post-infarction myocardial angiogenesis is impaired by DC depletion. A, Western

blotting images for VEGF level in the infarcted myocardium 7 days after MI. B, Quantification

data of VEGF based on immnoblotting experiment in control and DC-ablated mice (n = 4 per

group). C, Representative immunohistochemical staining for CD31 (red), Ki67 (brown) and

nuclei (blue) 7 days post-MI in control and DC-ablated mice. Scale bars indicate 30 m. D,

Quantitative assessment for blood vessels and CD31+ Ki67+ cells in control and DC-ablated

infarcts 7 days after MI (n = 4 per group). *P < 0.05 and **P < 0.01 vs sham of same group; †P <

0.05 and ††P < 0.01 vs control MI.

Figure 6. Enhanced infiltration of inflammatory M1 macrophages and diminished recruitment of

anti-inflammatory M2 macrophages by DC-ablation after MI. A, Representative photographs of

immunohistochemical staining for Mac3+ macrophages in infarcted and border areas 7 days after

MI. Scale bars indicate 100 m. B, Time course of Mac3+ macrophage infiltration in control and

DC-ablated mice after MI. Data were obtained from 5 independent experiments at each time

point. C, Representative flow cytometric dot plots of CD45+ leukocytes in the infarcted hearts

for CD206-Alexa 647 and F4/80-PE in control and DC-ablated mice 7 days after MI or sham

operation. D, The percentage of heart-infiltrating F4/80+ CD206- M1 (upper) and F4/80+ CD206+



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M2 (lower) macrophages in total F4/80+ macrophages of control and DC-ablated hearts 7 days

after MI or sham operation (n = 4 per group). E and F, Time course of MPO+ neutrophil (E) and

CD3+ T cell (F) recruitment after MI in control and DC-ablated mice analyzed by

immunohistochemical staining. Data were obtained from 5 independent experiments at each time

point. *P < 0.05 and **P < 0.01 vs sham of same group; †P < 0.05 vs control MI.

Figure 7. Increased Ly6Chigh monocyte but decreased Ly6Clow monocyte infiltration into the

infarcted myocardium in DC-ablation mice. A and B, Temporal changes of counts of peripheral

Ly6Chigh inflammatory (A) and Ly6Clow reparative (B) monocytes based on flow cytometric

analysis in control and DC-ablated mice. Data were obtained from 5 independent experiments at

each time point. C, Representative flow cytometric dot plots for Ly6C-APC and CD11b-PE after

sorting of CD45+ CD11bhigh cells from infarcted heart in control and DC-ablated mice 7 days

after MI or sham operation. D, Flow cytometric evaluation of heart-infiltrating Ly6Chigh (upper)

and Ly6Clow (lower) monocytes in control and DC-ablated mice 7 days after MI or sham

operation (n = 5 per group). *P < 0.05 and **P < 0.01 vs sham of same group; †P < 0.05 vs

control MI.



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Tsutomu Yoshikawa, Yasunori Okada, Shigeo Koyasu, Satoshi Ogawa and Keiichi FukudaKaneko, Yasuo Sugano, Toshiyuki Takahashi, Hitoshi Abe, Satsuki Mochizuki, Motoaki Sano, Atsushi Anzai, Toshihisa Anzai, Shigenori Nagai, Yuichiro Maekawa, Kotaro Naito, Hidehiro

Regulatory Role of Dendritic Cells in Post-Infarction Healing and Left Ventricular Remodeling

Print ISSN: 0009-7322. Online ISSN: 1524-4539 Copyright © 2012 American Heart Association, Inc. All rights reserved.

is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Circulation published online February 3, 2012;Circulation.

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SUPPLEMENTAL MATERIAL

Supplemental Methods

Bone-marrow (BM) Cell Preparation and BM Transplantation (BMT)

A BMT model was used to evaluate the role of dendritic cell (DC) by depleting CD11c+

diphtheria toxin receptor (DTR)+ cells by diphtheria toxin (DT) administration.

1 To harvest

BM cells, femurs and tibias were taken from 8-week-old male CD11c-DTR/GFP transgenic

mice. BM was flushed out with a 24-gauge syringe, and a single cell suspension in phosphate

buffered saline (PBS) was made through a 100-µm nylon mesh after red blood cell lysis using

ACK lysis buffer solution (0.15 M NH4Cl, 1.0 mM KHCO3, 0.1 mM Na2EDTA [pH 7.2]).

Five million BM cells were transferred intravenously into lethally irradiated (10 Gy) 6- to

8-week-old male wild-type (WT) C57BL/6 mice. The mice were allowed to rest for 8 weeks,

and the success of BMT was checked by polymerase chain reaction (PCR) to confirm the

presence of transgene in peripheral blood cells of the recipient mice before use. To evaluate

the BM chimerism after BMT, lethally irradiated (10Gy) CD45.1+ B6.SJL recipient mice

were reconstituted with BM cells of CD11c-DTR/GFP donor mice (CD45.2+) under the same

protocols described above.

Creation of Myocardial Infarction (MI)

Experimental MI was induced as previously described.2, 3

In brief, mice were anesthetized

2

with an intraperitoneal injection of ketamine (80 mg/kg) and xylazine (10 mg/kg), then

intubated and connected to a rodent ventilator. The chest cavity was opened via left

thoracotomy to expose the heart, and the left coronary artery was permanently ligated with a

7-0 silk suture at the site of its emergence from the left atrium. Complete occlusion of the

vessel was confirmed by the presence of myocardial blanching in the perfusion bed. Mice

that died within 24 hours after surgery were excluded from the experiment. Sham-operated

animals in both groups underwent the same procedure without coronary artery ligation. The

deceased mice were performed an autopsy to determine the cause of death: cardiac rupture

was confirmed by the presence of blood coagulation around the pericardial sac and in the

chest cavity, and heart failure was diagnosed by lung congestion with chest fluid

accumulation.

Echocardiography and Hemodynamics

Echocardiographic and hemodynamic evaluations were performed on day 28 after the

operation. For echocardiography, mice were anesthetized with 1-2% of isoflurane. M-mode

tracings were recorded through the anterior and posterior left ventricular (LV) walls at the

papillary muscle level to measure anterior wall thickness (AWT), posterior wall thickness

(PWT), LV end-diastolic diameter (LVEDD), and end-systolic diameter (LVESD), using an

echocardiographic system (12-MHz linear transducer; EnVisor C, Philips Medical Systems,

3

Andover, MA). The following formulas were used to calculate LV fractional shortening (FS)

and LV mass: FS (%) = [(LVEDD-LVESD)/LVEDD] × 100 and LV mass = 1.055 ×

[(AWT+PWT+LVEDD)3-LVEDD

3] , respectively.

Cardiac catheterization studies were performed using a 1.4 French microtip catheter

(SPR-671, Miller Instruments, Houston, TX) under sedation with intraperitoneal injection of

pentobarbital sodium (40 mg/kg) with spontaneous respiration. LV end-systolic pressure,

maximum rate of isovolumic pressure development and minimum rate of isovolumic pressure

decay were measured using analysis software (PowerLab, AD Instruments, Bella Vista, NSW,

Australia). Ten sequential beats were averaged for each measurement.

Morphometric Analysis

Heart tissue was fixed in formalin, embedded in paraffin, and cut into 5-µm-thick sections.

Sections were stained with hematoxylin and eosin, Masson’s trichrome and picrosirius red to

determine the infarct size and cardiac fibrosis, and identify their morphology. To evaluate the

quality of collagen fibers, picrosirius red stained-sections were studied with polarized

microscopy. The infarct size was assessed as total infarct circumference divided by total LV

circumference times 100, as described previously.4 The wall thickness of the scar at the

papillary muscle level was also measured. The fraction of collagen volume was assessed in

10 randomly chosen high-power fields (×200) in each section. These data were analyzed

4

using ImageJ software (version 1.38×, National Institutes of Health).

Real-Time Quantitative PCR

Total RNA was isolated by the acid-phenol extraction method in the presence of chaotropic

salts (Trizol, Invitrogen, Carlsbad, CA) and subsequent isopropanol-ethanol precipitation as

described previously.5 Reverse transcription was performed using a Super-Script First-Strand

Synthesis System (Invitrogen) in accordance with the manufacturer’s protocol. The

sequences of primer pairs were designed using Primer Express III software (Applied

Biosystems, Foster City, CA) and are described in online-only Data Supplement Table 1.

Real-time quantitative PCR was performed using an ABI Prism 7500 sequence detection

system (Applied Biosystems). The obtained data were normalized using the expression levels

of mouse 18S rRNA.

Western Blotting

Frozen tissue was homogenized in T-PER Tissue Protein Extraction Reagent (Thermo

Scientific, Rockford, IL) with protease inhibitors (Thermo Scientific). After centrifugation,

the supernatant was collected and protein concentration measured using a Bradford assay

system. Equal amounts of protein samples (30 µg) were electrophoresed on 4-12% NuPAGE

Bis-Tris gels (Invitrogen), and then proteins were electroblotted onto polyvinylidene

5

difluoride membranes. After blocking with 5% nonfat dried skim milk in Tris-buffered saline

(TBS) containing 0.1% Tween-20 at room temperature (RT) for 1 hour, the membranes were

incubated with the primary antibodies anti-interleukin (IL)-10 (Abcam, Cambridge, MA),

anti-inducible nitric oxide synthase (iNOS) (BD Bioscience, San Jose, NJ), anti-vascular

endothelial growth factor (VEGF) (Santa Cruz Biotech., Santa Cruz, CA) and

anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Santa Cruz Biotech.) at 4°C

overnight or for 1 hour at RT, followed by exposure to the secondary horseradish peroxidase

(HRP)-conjugated antibody for 1 hour at RT. The immunoblots were developed by enhanced

chemiluminofluorescence method. The signals were scanned with MF-ChemiBIS (DNR

Bio-Imaging Systems, Jerusalem, Israel) and analyzed using Quantity One software (Bio-Rad,

Hercules, CA).

Gelatin Zymography

To evaluate the activity of gelatinase, matrix metalloproteinase (MMP)-9 and MMP-2, gelatin

zymography was performed. Equal volumes of 30 µg protein were loaded onto each lane of

10% gelatin zymogram gels (Novex, Invitrogen). After running at 80V for 2 hours, the gels

were incubated in 2.5% Triton-X 100 for 30 min at RT, washed and then further incubated in

50 mM Tris-HCl, pH 7.4, containing 200 mM NaCl and 5 mM CaCl2 at 37°C overnight with

gentle agitation. After washing with deionized water, the gels were stained with Coomassie

6

blue for 90 min followed by destaining with a solution containing 30% ethanol and 10%

acetic acid. The presence of different MMPs was identified on the basis of their molecular

weight. The gels were photographed using MF-ChemiBIS (DNR Bio-Imaging Systems) and

analyzed using Quantity One software (Bio-Rad).

Immunohistochemical Staining

Immunohistochemical studies were performed by immunoperoxidase methods using

paraffin-embedded tissue sections. After inhibiting endogenous peroxidase activity, the

sections were incubated with primary anti-Mac3 (BD Bioscience), anti-MPO (Dako, Glostrup,

Denmark) or anti-CD3 (Dako) antibodies at 4°C overnight. After incubation, a Vectastain

ABC elite Kit (Vector Laboratories, Burlingame, CA) was used according to the

manufacturer’s instructions. Following visualization with 3,3’-diaminobenzidine, the sections

were finally counterstained with hematoxylin. The same methods were performed without the

primary antibodies, as negative controls. The numbers of Mac3+ macrophages, MPO

+

neutrophils, and CD3+ T lymphocytes were assessed by counting the total cell numbers in the

infarcted and border areas in 20 randomly chosen fields in each section. For the assessment

of endothelial proliferation in the infarcted heart, double immnohistochemical staining for

CD31 (Santa Cruz Biotech.) and Ki67 (Dako) was performed. After antigen retrieval, tissue

sections were incubated with primary antibodies followed by the reaction with secondary

7

antibodies and labeling with alkaline phosphatase (for CD31) and 3,3’-diaminobenzidine (for

Ki67). Finally, sections were counterstained with hematoxylin. The number of blood vessels

and CD31+ Ki67

+ proliferating endothelial cells were counted per unit area.

Immunofluorescent Staining

For immunofluorescent staining, frozen tissue sections were used. The excised hearts were

embedded in OCT compound, snap frozen in liquid nitrogen, and then cut into 6-µm-thick

sections. After fixing in acetone at RT for 10 min, cryosections were blocked using Block

Ace (DS Pharma Biomedical Co. Ltd., Osaka, Japan) at 37°C for 30 min, and then incubated

with anti-CD11c-APC (BD Bioscience) and purified anti-GFP (Medical & Biological

Laboratories Co. Ltd., Nagoya, Japan) antibodies at 4°C overnight to identify BM-derived

DCs. Alexa-Fluor 488-conjugated secondary antibody (Molecular Probes, Carlsbad, CA) was

added to detect GFP+ cells. To evaluate infiltrating leukocytes, primary purified anti-CD45

(BD Bioscience) and Alexa-Fluor 647-conjugated secondary (Molecular Probes) antibodies

were used. Nuclei were identified with DAPI. As negative controls, the same procedures

were performed without the primary antibodies. The sections were finally photographed

under a confocal laser-scanning microscope (TCS-SP5, Leica).

Preparation of Splenic and Peripheral Blood Cells

8

Spleens were removed, mushed and then passed through a 100-µm nylon mesh in PBS. After

addition of ACK lysis buffer solution to exclude erythrocytes, the single cell suspension in

PBS was refiltered through a 100-μm nylon mesh to remove connective tissue. Peripheral

blood was drawn via cardiac puncture and collected into a heparinized tube. ACK lysis buffer

solution was also added to the collected peripheral blood, followed by washing in PBS.

Isolation of Infiltrating Leukocytes from the Infarcted Heart

On the indicated days after the operation, mice were deeply anesthetized and intracardially

perfused with ice-cold PBS to remove blood cells before euthanasia. Infarcted tissue was

dissected, minced with fine scissors, and enzymatically digested with a cocktail of type II

collagenase (Worthington Biochemical Corporation, Likewood, NJ), elastase (Worthington

Biochemical Corporation) and DNase I (Sigma, St. Louis, MO) for 1 hour at 37°C with

gentle agitation. After digestion, the tissue was then triturated and passed through a 100-μm

cell strainer. For the separation of leukocytes, cells were incubated with biotin-conjugated

anti-CD45 antibody (BioLegend, San Diego, CA) at 4°C for 10 min, followed by incubation

with streptoavidin-conjugated microbeads (Miltenyi Biotec, Sunnyvale, CA) at 4°C for 15

min. Then, CD45+ leukocytes were positively collected by magnetic sorting with AutoMACS

(Miltenyi Biotec). The purity of cells was generally > 97%.

9

Flow Cytometry

Isolated splenocytes, peripheral blood cells, and leukocytes isolated from the heart were

analyzed by flow cytometry. To block nonspecific binding of antibodies to Fcγ receptors,

isolated cells were first incubated with anti-CD16/32 antibody (BD Bioscience) at 4°C for 5

min. Subsequently, the cells were stained with a mixture of the following antibodies at 4°C

for 20 min: anti-CD11c-PE, anti-CD11c-APC, anti-CD45.1-PerCP-Cy5.5, anti-MHCII-PE,

anti-CD11b-PE, anti-Mac3-PE, anti-Ly6C-APC, anti-F4/80-PE, anti-CD4-PE, anti-CD8-PE,

anti-CD19-PE (BD Bioscience), CD45.2-APC (BioLegend), and Alexa-Fluor 647-conjugated

anti-CD206 antibody (eBioscience, San Diego, CA). Peripheral blood monocytes were

defined as SSClow

CD11bhigh

cells as previously described.6 After staining with

7-amino-actinomycin D (Sigma) to discriminate dead cells, flow cytometry was performed

on a FACSCalibur (BD Bioscience) and the data analyzed with FlowJo software (Tree Star,

Ashland, OR).

10

References

1. Zammit DJ, Cauley LS, Pham QM, Lefrancois L. Dendritic cells maximize the

memory CD8 T cell response to infection. Immunity. 2005;22:561-570.

2. Katada J, Meguro T, Saito H, Ohashi A, Anzai T, Ogawa S, Yoshikawa T. Persistent

cardiac aldosterone synthesis in angiotensin II type 1A receptor-knockout mice after

myocardial infarction. Circulation. 2005;111:2157-2164.

3. Takahashi T, Anzai T, Kaneko H, Mano Y, Anzai A, Nagai T, Kohno T, Maekawa Y,

Yoshikawa T, Fukuda K, Ogawa S. Increased C-reactive protein expression

exacerbates left ventricular dysfunction and remodeling after myocardial infarction.

Am J Physiol Heart Circ Physiol. 2010;299:H1795-1804.

4. Kaikita K, Hayasaki T, Okuma T, Kuziel WA, Ogawa H, Takeya M. Targeted deletion

of CC chemokine receptor 2 attenuates left ventricular remodeling after experimental

myocardial infarction. Am J Pathol. 2004;165:439-447.

5. Sugano Y, Anzai T, Yoshikawa T, Maekawa Y, Kohno T, Mahara K, Naito K, Ogawa S.

Granulocyte colony-stimulating factor attenuates early ventricular expansion after

experimental myocardial infarction. Cardiovasc Res. 2005;65:446-456.

6. Sunderkotter C, Nikolic T, Dillon MJ, Van Rooijen N, Stehling M, Drevets DA,

Leenen PJ. Subpopulations of mouse blood monocytes differ in maturation stage and

inflammatory response. J Immunol. 2004;172:4410-4417.

11

Supplemental Table 1. Primers for real-time PCR in this study

Gene Sequence

IL-1 (F)TTGGGCCTCAAAGGAAAGAAT

(R)TGGGTATTGCTTGGGATCCA

TNF- (F)GCCAACGGCATGGATCTC

(R)GCAGCCTTGTCCCTTGAAGAG

IL-18 (F)CTGAAGAAAATGGAGACCTGGAA

(R)TCCGTATTACTGCGGTTGTACAGT

IL-10 (F)GCCAAGCCTTATCGGAAATG

(R)GGGAATTCAAATGCTCCTTGAT

CCL2 (F)CCTGGATCGGAACCAAATGA

(R)ACCTTAGGGCAGATGCAGTTTTA

CX3CL1 (F)TCCCCATCTGCCCTGACA

(R)AGTTCCAAAGCAAGGTCTTCCA

18S (F)TCGGAACTGAGGCCATGATT

(R)CCTCCGACTTTCGTTCTTGATT

(F), Forward Primer; (R), Reverse Primer.

Supplemental Figure Legends

Supplemental Figure 1. Evaluation of BM chimerism of BMT mice. A, Representative flow

cytometric dot plots of splenocytes for recipient-type CD45.1-PerCP-Cy5.5 and donor-type

CD45.2-APC. B, Representative flow cytometric dot plots of splenocytes for CD11c-PE and

CD45.2-APC, and histogram for GFP of CD45.2+ CD11c

+ splenocytes.

Supplemental Figure 2. Depletion of DCs by DT injection. A, Representative flow

cytometric dot plots of splenocytes for CD11c-APC and GFP or MHCII-PE in control and

DC-ablated mice. B, Representative flow cytometric histograms of splenocytes for CD4,

CD8, CD19 and CD11b in control (solid line) and DC-ablated (dotted line) mice.

Supplemental Figure 3. Inefficacy of DT on the WT mice heart. Echocardiographic

measurements and heart/lung weight adjusted for body weight in non-treated and DT-treated

WT mice are shown.

Supplemental Figure 4. Enhanced infiltration of CD45+ leukocytes and Mac3

+ macrophages

into the infarcted heart by DC-ablation. A, Time course of CD45+ leukocyte infiltration into

the heart of control and DC-ablated mice after MI. Data were obtained from 5 independent

experiments at each time point. B, Representative immunofluorescent staining patterns for

13

CD45 in infarcted and border areas 7 days after MI. Scale bars indicate 30 μm. C,

Representative flow cytometric dot plots of heart-infiltrating CD45+ leukocytes for Mac3-PE

in control and DC-ablated mice 7 days after MI. D, Quantification of Mac3+ macrophages

based on flow cytometry in control (n = 5) and DC-ablated (n = 5) mice 7 days after MI. *P <

0.05 and **

P < 0.01 vs sham of same group; †P < 0.05 and

††P < 0.01 vs control MI.

Supplemental Figure 5. DC-ablation induces marked monocyte recruitment after MI. A,

Temporal changes in number of total peripheral blood monocytes by flow cytometry. B,

Number of tissue monocytes in control and DC-ablated mice 7 days after MI or sham

operation. *P < 0.05 and

**P < 0.01 vs sham of same group;

†P < 0.05 vs control MI.

14

The online data supplement Figure 1.

A

B

97.4%

GFP

CD45.1

CD

45

.2

96.2%

2.9%

CD11c

CD

45

.2

15

A

B

CD11c

GFP

CD

11

cM

HC

cla

ss II

Control

0.73%

DC-ablation

0.01%

0.65% 0.02%

CD4

Co

un

t

CD8 CD19 CD11b


16

0

2

4

6

LV

ED

D (

mm

)

WT WT+DT WT WT+DT

Sham MI

P = 0.68

P = 0.86

0

1

2

3

4

5

LV

ES

D (

mm

)WT WT+DT WT WT+DT

Sham MI

P = 0.96

P = 0.75

0

2

4

6

8

10

LW

/BW

(m

g/g

)

WT WT+DT WT WT+DT

Sham MI

P = 0.94

P = 0.45

0

1

2

3

4

5

HW

/BW

(m

g/g

)

WT WT+DT WT WT+DT

Sham MI

P = 0.73

P = 0.83

0

20

40

60

FS

(%

)

WT WT+DT WT WT+DT

Sham MI

P = 0.64

P = 0.81


17


A B


DAPI / CD45

Infa

rct

are

aB

ord

er

are

a


0

20

40

60

80

Control

DC-ablation

CD

45

+ leu

ko

cyte

s (

10

4)

†

**

**

*

Unstained

Ma

c3

Control MI

9.25%

DC-ablation MI

15.30%

0

5

10

15

20M

ac3

+ m

acro

ph

ag

es (

%)

Control

MIDC-ablation

MI

††

C D

18

A

** *

†

**

*


0

5

10

15

Control

DC-ablation

Pe

rip

he

ral b

loo

d m

on

ocyte

s (

%)

B

0

2

4

6

8

Tis

su

e m

on

ocyte

s (

%)

controlDC-

ablationcontrol

DC-

ablation

Sham MI

†

**

*


regulatory role of dendritic cells in post-infarction ...€¦ · 3 introduction left ventricular...

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