Interferon Regulatory Factor 5 Controls Necrotic Core Formation in Atherosclerotic Lesions by Impairing Efferocytosis

Supplemental Digital Content is available in the text.


Measurement of lesion formation in perivascular cast-induced injury
Nine weeks following cast placement, mice were euthanized, terminal blood collected via cardiac puncture and the vasculature perfused with 0.9% w/v saline. The injured carotid was dissected out and frozen at -80 o C in Optimal cutting temperature (OCT) compound (ThermoScientific, Runcorn, UK). Serial 5μm cryosections were taken through the entire length of the carotid artery. Staining of the elastic lamina was performed using the Accustain kit (Sigma-Aldrich Inc., St. Louis, USA) according to the manufacturer's instructions. Measurement of lesion and vessel areas was performed using ProgRes CapturePro image analysis software (version 2.5.2.0, Jenoptik, Germany). The area between the internal and external elastic lamina was taken as the medial area and the intimal area was calculated by subtracting the lumen area from the internal elastic lamina area. The intimal medial ratio (IMR) was then calculated by dividing the intimal area by the medial area.

Hematoxylin and Eosin (H+E) staining and necrosis quantification
H+E staining was performed using a Tissue Tek Prisma/Film automated slide stainer (Sakura, Japan). Slides were fixed in 10% buffered formalin for 10 minutes, rinsed in distilled water and then stained with Harris hematoxylin for 8 minutes, washed in running tap water for 5 minutes, and differentiated with 0.3% acid alcohol for 2 minutes and rinsed in distilled water. The slides were counterstained with eosin for 2 minutes before being rinsed in distilled water. Necrotic areas (defined as being H+E free) were quantified using Clemex Vision Lite version 5.0. Only areas larger than 3000µm 2 in the aortic root and 300µm 2 in the carotid were included in the analysis.
Absolute values were obtained by calibrating the software using an image of a micrometer slide taken at the same magnification. Necrotic lesion area was calculated by dividing the necrosis area by the lesion area and expressing it as a percentage.

Murine Immunohistochemistry
Immunohistochemistry was performed on 5μm cryosections using standard avidin biotinylated enzyme complex (ABC) methods as previously published 2 . In brief, sections were fixed in ice-cold acetone before incubation with 10% normal rabbit or goat serum for one hour. Following a wash in PBS, endogenous avidin and biotin were blocked using Vector avidin/biotin blocking kit (Vector labs, Peterborough, UK) according to manufacturer's instructions. Sections were then incubated with primary antibodies against CD11c (BD Biosciences 2.5µg/mL), CD68 (AbD Serotec, 5µg/ml), iNOS (Abcam, 1µg/mL), CD206 (AbD Serotec, 5µg/mL), IRF5 (Abcam, 0.5µg/mL) or HO-1 (Abcam, 5µg/mL) for 45 minutes at room temperature, followed by relevant biotinylated secondary antibodies. Following blocking of endogenous peroxidase activity with 0.3% hydrogen peroxide, sections were incubated with avidin and biotinylated horseradish peroxidase macromolecular complexes using Vectastain Elite ABC kit (Vector Labs) according to manufacturer's instructions. Bound peroxidise was detected using 3,3'-diaminobenzidine (DAB) and nuclei counterstained with hematoxylin. Staining using an appropriate isotype-matched control was performed on a consecutive section as a control. Smooth muscle cell staining was performed using an antibody against alpha smooth muscle actin conjugated to Cy3 (Sigma 5µg/mL) and DAPI counterstaining of nuclei.

Immunofluorescence staining
Dual staining of aortic root sections with antibodies against IRF5 and CD68, CD11c or a-smooth muscle actin was performed using tyramide signal amplification (TSA) using a biotin-TSA kit as per manufacturer's instructions (Perkin Elmer). In brief, 5µm cryosections were fixed in ice-cold acetone for 5 minutes before endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide in PBS for 15 minutes.
Endogenous avidin and biotin activity were then blocked using the Vector avidin/biotin blocking kit. Following blocking with 20% normal goat serum for one hour, sections were blocked with TNB blocking buffer (0.1M Tris-HCl pH 7.5, 0.15M NaCl, 0.5% blocking buffer as supplied in kit) for 30 minutes before incubation with an antibody against IRF5 (Abcam, 0.5μg/mL) for 45 minutes. Following washing, sections were incubated with relevant biotinylated secondary antibodies then incubated with streptavidin-HRP for 30 minutes. Biotinyl tyramide working solution was then added for 5 minutes followed by streptavidin-Alexa Fluor 488 (Life Technologies). Sections were then re-blocked and stained with an antibody against CD68 (AbD Serotec, 5μg/mL) or CD11c (AbD Serotec 20μg/mL) and an Alexa Fluor 568 conjugated secondary antibody and DAPI (Life technologies). Alternatively, slides were stained with an antibody against alpha smooth muscle actin conjugated to Cy3 (Sigma 5µg/mL) and DAPI. Slides were viewed on an Ultraview confocal microscope (PerkinElmer Life Sciences, Cambridge, UK).

Quantification of murine immunohistochemical staining
For all quantification, images were captured under identical microscope, camera and light conditions, coded and analyzed blind. Aortic root lesion area staining positive for a given marker was quantified using Clemex Vision Lite version 5.0. Using the image analysis software, positive staining was detected and lesion area measured.
Absolute values were obtained by calibrating the software using an image of a micrometer slide taken at the same magnification. Lesion area fraction staining positive was calculated by dividing the area staining positive by the lesion area and expressing it as a percentage.

Analysis of serum cholesterol
Total serum cholesterol levels were measured enzymatically using Infinity Total Cholesterol (Thermo Scientific), according to the manufacturer's instructions. A calibration serum (Randox Laboratories) with a known cholesterol concentration was used as a reference.

In situ efferocytosis assay
Sections of cast-induced carotid lesions were stained for apoptosis and DNA using the BrdU-Red DNA Fragmentation kit (TUNEL, ab66110 Abcam, Cambridge, UK) according to manufacturer's instructions. Slides were counterstained for macrophages using an antibody against CD68 (Alexa Fluor 647, AbD Serotec). The most stenotic part of each section was imaged using an Olympus FV1200 IX83 confocal system. Images were analyzed using ImageJ (1.50I, NIH, USA) and Photoshop CS6 (13.0.1x32, Adobe Systems Inc, USA). TUNEL+ apoptotic cells surrounded by a CD68+ cell were counted as cells undergoing efferocytosis, as previously described 3 . TUNEL+ apoptotic cells not surrounded by a CD68+ cell were counted as apoptotic cells not undergoing efferocytosis. All analysis was performed by 2 independent assessors blinded to the experiment details.

Transfection of murine GMCSF Macrophages with RNAi Oligos
Bone marrow macrophages were generated as above and were then plated at 1 x 10 6 in a 12 well plate with 20ng/ml fresh GMCSF overnight at 37 0 C. The next day, the oligo (ON-TARGET plus SMART pool siRNA for mouse Mfge8, Itgb3 or a nontargeting control, Dharmacon), Dharmafect 1 (Dharmacon) and Opti-mem (Gibco) mix was prepared to give a final concentration of 100nM of oligo and left to incubate 20min at RT. The macrophage growth media was then replaced with the relevant oligo-mix (in serum-free RPMI) and incubated at 37 0 C for 2hrs. After removal of the transfection complexes, 1ml of RPMI with 5%FCS was added to the well and the plate incubated for 48hrs at 37 o C. The efferocytosis assay was then performed as described.

Measurement of Mfge8 release
Supernatants from GMCSF-derived bone marrow cultures were removed after one week and stored at -80 o C for batch analysis. A commercially available mouse MFGE8 ELISA kit was used (Biolegend) in accordance with the manufacturer's instructions. Samples were run in duplicate.

Real time quantitative PCR
Total RNA was extracted from GMCSF matured macrophages, aorta and PALN from 20 week old ApoE -/and ApoE -/-Irf5 -/mice using a Qiagen RNeasy mini kit, according to manufacturer's instructions. Total RNA was reverse transcribed to cDNA using a High Capacity Reverse Transcription Kit (Life Technologies). Following preamplification (14 cycles), RT-PCR was performed using either custom TaqMan Array microfluidic cards or individual TaqMan Gene Expression Assays and TaqMan universal PCR Master Mix (Life technologies) on an ABI 7900HT fast real-time PCR system (Applied Biosystems). PCR amplification was carried out for 40 cycles. All samples were analysed in triplicate and were normalized to 18s. The 2-ΔΔCt method was used to analyse the relative changes in gene expression.

Chromatin immunoprecipitation and next-generation sequencing
The IRF5 ChIP-seq analysis were performed as previously described (Accession number: E-MTAB-2661) 6 . 300 million GMCSF matured bone marrow derived macrophages from wild type and Irf5 -/mice were used for IRF5 ChIP following stimulation with LPS for 0 and 2hrs in duplicate. Cells were fixed for 10 minutes with 1% formaldehyde, quenched with 125mM of Tris pH7.5 and washed with ice-cold PBS. Nuclear lysates were isolated as previously described and sonicated with a Bioruptor (Diagenode) to obtain chromatin fragment sizes that average 300bp. Each lysate was immunoprecipitated with 10μg of IRF5 antibody (Abcam; ab21689). ChIP was performed as described previously. ChIPped DNA was quantified with the Quant-iT dsDNA High Sensitivity Assay Kit (Invitrogen #Q33120). DNA yields ranged from 10-20 ng. The ChIP-Seq datasets were generated using 50bp paired end sequencing. Reads were trimmed using Trimmomatic, and mapped to the mm10 genome using Bowtie2. Before peak calling with MACS2 using the following settings: --mfold 10 30 --gsize mm --qvalue 0.05. Called peaks in the WT samples were then filtered against those in Irf5 -/to exclude any false positives. Read coverage profiles were computed with 25bp resolution and normalized by reads per kilobase per million mapped reads (RPKM) using deepTools. Called peaks and read coverage profiles were visualized using IGV.

Human carotid plaque sample preparation and histology
Human carotid plaques from the Carotid Plaque Imaging Project (CPIP) biobank (Malmö, Sweden) were analyzed. Plaques were collected at carotid endarterectomy and the indications for surgery were plaques associated with ipsilateral symptoms (Transitory ischemic attack, stroke or amaurosis fugax) and >70% stenosis, measured by duplex, or plaques not associated with symptoms and stenosis >80%.
After surgical removal, plaques were snap-frozen in liquid nitrogen. Fragments