Courties et al.: RNAi silencing in wound macrophages
SUPPLEMENTARY METHODS
Animal models. C57BL/6J and B6.129P2-Apoetm1Unc/J (ApoE-/-) mice used in this study were
purchased from Jackson Laboratory. ApoE-/- mice were fed on a high-cholesterol diet for 6
months (Harlan Teklad, 0.2% total cholesterol). Myocardial infarction was induced by
permanent coronary ligation (1). Briefly, mice were intubated and ventilated with 2% isoflurane
supplemented with oxygen. Thoracotomy was performed in the fourth left intercostal space. The
left coronary artery was identified and permanently ligated with a monofilament nylon 8-0
suture. The thorax was closed with glue. The skin wounds were induced as described (2). In
brief, the dorsal region of anesthetized mice was shaved using animal clipper and a depilatory
agent and skin was swabbed with alcohol. Wounds were patterned using a sterile 6-mm punch
biopsy tool (Miltex). Skin was excised with sterile surgical scissors and donut-shaped stents of
silicone sheet were sutured around the biopsy. The studies were approved by the Subcommittee
on Animal Research Care at Massachusetts General Hospital (13th Street, Charlestown, MA).
Synthesis and screening of IRF5 siRNAs. Twenty-four siRNAs duplexes were generated
against the murine IRF5 transcript (accession number NM_012057.3). Single-strand RNAs were
designed and produced at Alnylam Pharmaceuticals as described (3). The mouse macrophage
cell line J774A.1 was transfected with either siRNAs targeting the mouse IRF5 gene or with nontargeting control siRNA complexed with Lipofectamine® RNAiMAX Transfection Reagent
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Courties et al.: RNAi silencing in wound macrophages
(Invitrogen) at 0.1 nM and 10 nM final concentrations. Cells were harvested at 24 hours after
transfection and expression levels of IRF5 mRNA were quantified by qRT-PCR using TaqMan
primers. The following sequence of IRF5 siRNA was scaled up for in vivo studies : 5’cuGcAGAGAAuAAcccuGAdTsdT-3’ (sense), 5’-UcAGGGUuAUUCUCUGcAGdTsdT-3’
(antisense) wherein lower case letters identify 2’OMe modified nucleotides. Nanoparticle
encapsulation was done as described previously (4, 5).
Intravenous injection of siRNAs. Mice were anesthetized and injected through the tail vein
with 0.5 mg/kg of either siIRF5 or with control siRNA targeting luciferase (siCON). For
biodistribution studies, mice were injected with 1 mg/kg of Alexa Fluor-647 labeled siRNA lipid
nanoparticles.
Cell isolation and flow cytometry. To obtain single-cell suspensions from heart tissue, infarcts
were excised, minced with a fine scissor prior to digestion in 450 U/ml collagenase I, 125 U/ml
collagenase XI, 60 U/ml DNase I and 60 U/ml hyaluronidase (Sigma-Aldrich) for 1 hour at 37°C
under agitation (750 rpm). Cells were then triturated through a 40µm nylon mesh (BD Falcon),
washed and centrifuged (8 min, 300 g, 4°C). Cells were first stained with a cocktail of
Phycoerythrin (PE) anti-mouse antibodies including CD90.2 (clone 53-2.1), CD19 (clone 1D3),
NK1.1 (clone PK136), Ly-6G (clone 1A8) and Ter-119 (clone TER-119). Cells were then
stained with anti-mouse CD11b (clone M1/70), F4/80 (clone BM8) and Ly6C (clone AL-21).
Neutrophils were identified as (CD90.2/CD19/NK1.1/Ly-6G/Ter119)high, CD11bhigh. Ly-6Chigh
monocytes were identified as (CD90.2/CD19/NK1.1/Ly-6G/Ter119)low, CD11bhigh, F4/80low, Ly-
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Courties et al.: RNAi silencing in wound macrophages
6Chigh. Macrophages were identified as (CD90.2/CD19/NK1.1/Ly-6G/Ter119)low, CD11bhigh,
F4/80high, Ly-6Clow.
Isolation of cells from the heart. Depletion of undesired lymphocytes and granulocytes was
performed using column-based magnetic cell sorting according to the manufacturer’s instructions
(Miltenyi Biotec). Briefly, single cell suspensions were stained using a cocktail of PE-conjugated
antibodies directed against CD90, CD19, NK1.1, Ter119 and Ly6G, followed by incubation with
anti-PE Microbeads. The enrichment was evaluated by flow cytometry isolated
monocytes/macrophages were used for downstream qRT-PCR and western blot analyses.
Quantitative RT-PCR. Total messenger RNA (mRNA) was extracted using the RNeasy Micro
Kit (Qiagen) according to manufacturer’s instructions. One microgram of mRNA was reverse
transcribed using the high capacity RNA to cDNA kit (Applied Biosystems). TaqMan gene
expression assays (Applied Biosystems) were used to quantify target genes. The relative changes
were normalized to Gapdh mRNA using the 2-∆∆CT method.
Western blots. Myeloid cells from heart tissue were isolated as described above, washed with
ice-cold PBS and homogenized on ice using RIPA lysis buffer (Millipore) supplemented with
complete protease inhibitor cocktail (Roche). Protein concentration was measured using BCA
assay (Pierce). Samples of 15 µg were loaded on 10% SDS-PAGE and transferred onto PVDF
membranes (Bio-Rad). Membranes were blocked with 5% non fat dry milk in TBS-Tween 0.1%
and incubated with antibodies against IRF5 (Abcam, ab21689), and peroxidase-coupled
antibodies as the secondary antibody (Abcam, ab6721). Gapdh or b-tubulin were used as
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Courties et al.: RNAi silencing in wound macrophages
controls. Signals were visualized with enhanced chemiluminescence detection system (ECL Plus,
Amersham Life Science), and densitometric analysis was performed with ImageJ 1.40g
(National Institutes of Health).
TTC staining. The hearts were excised under a microscope and were then cut into myocardial
rings of 1 mm thickness using a rodent heart slicer (Zivic Instruments), which were stained with
2-3-5-triphenyl tetrazolium chloride (TTC) to demarcate the infarct. Thereafter, the rings were
scanned (HP Scanjet 4300).
Fluorescence reflectance imaging (FRI). Heart slices were imaged using a planar fluorescent
reflectance imaging system (OV-110, Olympus) with an excitation wavelength 680 nm. Light
and near infrared fluorescence (NRIF) images were obtained with respective exposure times
between 75 ms and 60 seconds. Animals were maintained under a continuous flow of 1-2%
isoflurane in oxygen for maintenance of anesthesia throughout the imaging session.
Fluorescence Molecular Tomography-Computed Tomography (FMT/CT). FMT imaging
(680/700 nm excitation/ emission) was performed to investigate in vivo magnitude of
inflammation in infarct wounds (6, 7). Five nmoles of a pan-cathepsin protease sensor (Prosense680) were injected i.v. 24 hours before imaging. A quantitative 3D dataset in which fluorescence
per voxel was expressed in nM was reconstructed. FMT was followed by CT (Inveon PET-CT,
Siemens) to identify anatomic regions. Contrast-enhanced X-ray computed tomography localized
the infarcts. This anatomical information guided the placement of the volume of interest in the
quantitative protease activity map concomitantly obtained by hybrid FMT. An imaging cartridge
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Courties et al.: RNAi silencing in wound macrophages
containing the anesthetized mouse was placed into a custom machined Plexiglas holder that
supplies isoflurane during imaging. The CT x-ray source, with an exposure time of 370-400 ms,
was operated at 80 kVp and 500 μA. During CT, isovue-370 was infused continuously at 20
μL/min through a tail vein catheter. The CT reconstruction protocol performed bilinear
interpolation, using a Shepp-Logan filter, and scaled pixels to Hounsfield units. Image fusion
relied on fiducial markers and used Osirix software (The Osirix Foundation, Geneva).
Magnetic resonance imaging (MRI) In vivo MRI was performed on days 1 and 21 after
coronary ligation. Cine images of the left ventricular short axis were obtained using a 7 Tesla
horizontal bore Pharmascan (Bruker) and a custom-built mouse cardiac coil in birdcage design
(Rapid Biomedical). Acquisition was done as described previously, using an ECG triggered and
respiratory gated fast low angle shot sequence. The echo time was 2.7ms, and the flip angle 30
degrees (60 degrees for delayed enhancement imaging after intravenous injection of Gd-DTPA
for measurement of infarct size) (8). Image analysis was done using the software Segment
(http://segment.heiberg.se) (9).
Histology. To eliminate blood contamination, hearts were perfused with ice-cold PBS after mice
were euthanized. Hearts were removed, rinsed in PBS, embedded in O.C.T. compound (Sakura
Finetek), and frozen in an isopentane bath on dry ice. For immunofluorescence staining, sections
(5 μm) were either stained with anti-CD11b antibody (clone M1/70, BD Biosciences) followed
by a biotinylated anti-rat secondary antibody and fluorescein streptavidin (Vector Laboratories,
Inc.) or co-stained with the TUNEL reagents (DeadEnd Fluorometric TUNEL System, Promega)
and then incubated with a rat anti-mouse MAC-3 antibody. Streptavidin-DyLight594 (Vector
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Courties et al.: RNAi silencing in wound macrophages
Laboratories, Inc) was used to detect the MAC-3 antibody. The slides were cover slipped using a
mounting medium with DAPI (Vector Laboratories, Inc.) to identify nuclei. Images were
observed and captured using Nikon Eclipse 80i with a Cascade Model 512 B camera (Roper
Scientific). For immunohistochemistry, histology of the heart was performed on day 7 after MI
in ApoE-/- mice. Frozen sections (5 μm) were stained for neutrophils (NIMPR14, Santa Cruz
Biotechnology, Inc.), monocytes (CD11b, M1/70, BD Biosciences), macrophages (MAC-3,
M3/84 BD Biosciences), neovessels (PECAM-1, MEC13.3 BD Biosciences), and collagen
deposition (Collagen I, Abcam), and smooth muscle cells (a-SMA, Abcam). The appropriate
biotinylated secondary antibodies, ABC kit (Vector Laboratories, Inc.) and AEC substrate
(Dako) were used for color development, and all the sections were counterstained with Harris
hematoxylin. The slides were scanned by a digital slide scanner, NanoZoomer 2.0-RS in 40x
high resolution mode (Hamamatsu, Japan). The positive area was quantified using IPLab
(version 3.9.3; Scanalytics, Inc.) and analyzing five high power fields per section and per animal.
Statistics. Data are expressed as mean ± sem. Analyses were performed using Prism 6.0a
(GraphPad Software Inc.). The group means were compared using a t-test (for 2 groups) and
ANOVA, followed by Bonferroni post-tests (for > 2 groups). P values of <0.05 indicate
statistical significance.
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Courties et al.: RNAi silencing in wound macrophages
SUPPLEMENTARY DISCUSSION
Why do we detect nanoparticle delivery to infarct macrophages in non-reperfused MI while the
Evans blue method does not show dye distribution to the infarct in the same model?
The Evans blue method uses digital photography (10), and hence light of the visible spectral
wavelength, to detect poorly perfused myocardium downstream of the ligated coronary artery.
Other techniques such as MRI detection of delayed infarct enhancement after injection of GdDTPA (Fig. 6A in main manuscript) or fluorescence sensing of a near infrared fluorochrome
attached to siRNA (Fig. 2) do not rely on visible light. Near infrared imaging (11), near infrared
microscopy and near infrared detection of fluorochromes inside single cells by FACS (as used in
this paper, Fig. 2) are very sensitive techniques due to the high resolution, and because photons
with wavelength in the near infrared are absorbed to a lesser extent in myoglobin-rich
myocardium when compared to the visible light (12). In addition, fluorescence imaging uses
long acquisition times and post-acquisition signal amplification to increase sensitivity. We
hypothesize that if one would look for Evans blue in non-reperfused infarcts with a highly
sensitive technique one could detect this small molecule dye, albeit at a much lower level when
compared to non-infarcted myocardium.
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Courties et al.: RNAi silencing in wound macrophages
SUPPLEMENTARY REFERENCES
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Nahrendorf M, Swirski FK, Aikawa E et al. The healing myocardium sequentially
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Galiano RD, Michaels Jt, Dobryansky M, Levine JP, Gurtner GC. Quantitative and
reproducible murine model of excisional wound healing. Wound Repair Regen.
2004;12:485–92.
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Frank-Kamenetsky M, Grefhorst A, Anderson NN et al. Therapeutic RNAi targeting
PCSK9 acutely lowers plasma cholesterol in rodents and LDL cholesterol in nonhuman
primates. Proc Natl Acad Sci U S A. 2008;105:11915–20.
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Akinc A, Zumbuehl A, Goldberg M et al. A combinatorial library of lipid-like materials for
delivery of RNAi therapeutics. Nat Biotechnol. 2008;26:561–69.
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Leuschner F, Dutta P, Gorbatov R et al. Therapeutic siRNA silencing in inflammatory
monocytes in mice. Nat Biotechnol. 2011;29:1005–10.
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Nahrendorf M, Sosnovik DE, Waterman P et al. Dual channel optical tomographic imaging
of leukocyte recruitment and protease activity in the healing myocardial infarct. Circ Res.
2007;100:1218–25.
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Panizzi P, Swirski FK, Figueiredo JL et al. Impaired infarct healing in atherosclerotic mice
with Ly-6C(hi) monocytosis. J Am Coll Cardiol. 2010;55:1629–38.
8.
Yang Z, Berr SS, Gilson WD, Toufektsian MC, French BA. Simultaneous evaluation of
infarct size and cardiac function in intact mice by contrast-enhanced cardiac magnetic
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resonance imaging reveals contractile dysfunction in noninfarcted regions early after
myocardial infarction. Circulation. 2004;109:1161–67.
9.
Heiberg E, Sjogren J, Ugander M, Carlsson M, Engblom H, Arheden H. Design and
validation of Segment--freely available software for cardiovascular image analysis. BMC
Med Imaging. 2010;10:1.
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Gao E, Lei YH, Shang X et al. A novel and efficient model of coronary artery ligation and
myocardial infarction in the mouse. Circ Res. 2010;107:1445–53.
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Leuschner F, Nahrendorf M. Molecular imaging of coronary atherosclerosis and
myocardial infarction: considerations for the bench and perspectives for the clinic. Circ
Res. 2011;108:593–606.
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Weissleder R, Ntziachristos V. Shedding light onto live molecular targets. Nat
Med. 2003;9:123–28.SUPPLEMENTARY FIGURE LEGENDS
Supplementary figure 1: IRF5 siRNA treatment does not change IRF5 expression in
neutrophils and lymphocytes. MI was induced in ApoE-/- mice by coronary ligation. Mice were
injected daily for 4 days with 0.5mg/kg of siRNA silencing IRF5 (siIRF5). Control animals were
treated with control siRNA (siCON). FACS analysis shows IRF5 protein level in neutrophils and
lymphocytes. Representative histogram plots display control siRNA (red), IRF5 siRNA (blue)
and isotype control (grey). Bar graph shows mean fluorescence intensity (MFI) for IRF5 (n=8
per group).
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Courties et al.: RNAi silencing in wound macrophages
Supplementary figure 2: Chemokine and chemokine receptor expression in infarct tissue.
MI was induced in ApoE-/- mice by coronary ligation. Mice were injected daily for 4 days with
0.5mg/kg of siRNA silencing IRF5 (siIRF5). Control animals were treated with control siRNA
(siCON). qRT-PCR analysis of neutrophil (cxcl1 and cxcl2) and monocyte-related chemokines
(ccl2 and cx3cl1) and their receptors (ccr2 and cx3cr1) in infarct tissue, normalized to Gapdh
mRNA levels (n = 6 per group).
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Courties et al.: RNAi silencing in wound macrophages
Supplementary figure 3: IRF5 gene silencing does not change macrophage apoptosis in
infarct tissue. Hearts from ApoE-/- mice treated with siCON or siIRF5 were harvested on day 7
after MI. Sections were co-stained for MAC-3 (red), terminal deoxynucleotidyl transferasemediated dUTP-biotin nick end labeling (TUNEL; green) and DAPI (blue). (n=5 per group).
Scale bar indicates 50 µm.
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Courties et al.: RNAi silencing in wound macrophages
Supplementary figure 4: IRF5 silencing is associated with decreased matrix degradation
after myocardial infarction. MI was induced in ApoE-/- mice by coronary ligation. Mice were
injected daily for 4 days with 0.5mg/kg of siRNA silencing IRF5 (siIRF5). Control animals were
treated with control siRNA (siCON). qRT-PCR analysis of IRF5 (a), MMP (b) and TIMP gene
expression (c) in infarct tissue, relative to Gapdh mRNA levels (n=6 per group). (d) MMP:TIMP
expression ratios show lowered MMP-9:TIMP-1 ratio in IRF5 siRNA treated animals.
Mean±sem, * P < 0.05.
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