Animals and diet

8-week-old, female Ldlr–/– mice (B6.129S7-Ldlrtm1Her/J) were lethally irradiated (10 Gy, irradiator IBL637C g-ray 137Cs; Schering Cis-Bio International, France) and reconstituted with a 1:1 mixture of 3,000,000 bone marrow cells from CD45.1 C57Bl/6 (B6.SJL-Ptprca Pepcb/BoyJ) and CD45.2 (Msr1–/– [B6.Cg-Msr1tm1Csk/J], Cd36–/– [B6.129S1-CD36tm1Mfe/J], Myd88–/– [B6.129P2(SJL)-Myd88tm1Defr/J]) Lxrα/β−/−, Nlrp3–/– (B6N.129-Nlrp3tm2Hhf/J), Pycard–/– (B6.129-PycardtmTsc), Casp1–/– (B6-Casp1tm2Gross/N), Il-1r–/– (B6.129S7-Il1r1tm1lmx/J), LysMCre/WT:ABCA1/G1fl/fl (B6.129P2-Lyz2tm1(cre)Ifo/J, Mac-ABC-DKO, in-house breeding) mice, as previously described [31]. Mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). Following 6 weeks of reconstitution consuming chow diet, mice were fed a high-cholesterol diet (HCD) (1.25% w/w cholesterol, D12108 mod.; Ssniff GmBH, Soest, Germany) for 4 or 12 weeks to study early and advanced atherosclerosis, respectively. Mice were housed under specific pathogen-free conditions.

8-week-old Apolipoprotein E (Apoe)-deficient mice were sex-matched and randomly assigned to 2 groups. They were fed a cholesterol diet (21.2% fat by weight and 0.2% cholesterol, TD.88137, Envigo) for 12 weeks. Four weeks prior to euthanasia, mice received either placebo or MCC950 (weight-adjusted 10 µg/g) by intraperitoneal injection every two days. To visualize local macrophage proliferation, 125 µg 5-ethynyl-2-deoxyuridin (EdU) (ThermoFisher Scientific, Waltham, MA, USA) was injected intraperitoneally for the last three hours prior to sacrifice.

Human plaques

Tissue containing atherosclerotic plaques was collected from patients within 6 h after elective eversion carotid endarterectomy. All patients consented to take part in the study prior to surgery (Freiburg ethic approval no. 249/14). Residual blood and adventitial fat were removed from the specimens, and plaques were cut into 2 mm thick cross-sections. Three adjacent sections were transferred into 1 ml of medium each, with an EdU concentration of 50 µmol/L. Sections were either treated with MCC950 (1 µmol/L), with MCC950 (1 µmol/L) plus recombinant human interleukin-1 beta (IL-1β) (125 pg/ml), or with vehicle (control) for 24 h. After 24 h of treatment, the supernatants were collected and used to determine IL-1β concentrations. Tissues were embedded in Tissue-Tek OCT (Sakura Finetek, Torrance, CA, USA), frozen and cut into 10 µm slices. For staining, slices were fixed in cold acetone and permeabilized using 0.1% TritonX 100 solution (Applichem GmbH, Darmstadt, Germany); the Click-iT EdU Imaging Assay Kit (ThermoFisher Scientific, Waltham, MA, USA) was then used according to the manufacturer's protocol. For blocking, the sections were incubated with 5% goat serum and incubated with primary CD68 antibody (Clone KP1, Bio-Rad Laboratories, Hercules, CA, USA, 1:250) overnight. The secondary antibody (goat anti-mouse, 1:500; Goat anti Mouse IgG H&L AF647 preadsorbed; Abcam, Cambridge, UK) was added to the sections for 4 h. Finally, the sections were treated with the Vector TrueVIEW Autofluorescence Quenching Kit (Vector Laboratories, Burlingame, CA, USA) to reduce autofluorescence, and were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA).

Bone marrow-derived macrophages (BMDMs)

Bone marrow cells were extracted from femurs and tibiae. The cell suspension was processed through a 40 μm cell strainer. For macrophage differentiation, 105 bone marrow cells were cultured in 1 ml conditioned media (RPMI with 10% fetal calf serum (FCS); 1% nonessential amino acids (NEAA); 1% penicillin/streptomycin (PenStrep); 30 ng/ml murine macrophage colony stimulating factor (M-CSF; PeproTech, Hamburg, Germany)) per well in a 48-well plate for 5–7 days. The media, with supplements, was changed every two days.

Macrophage uptake of oxidized (ox)LDL or acetylated (ac)LDL

Murine BMDM were incubated with 20 μg/ml human DiI-labeled medium oxidized LDL or Dil-labeled acetylated LDL (DiI-oxLDL/ Dil-acLDL, Kalen Biomedical, Montgomery Village, MD, USA) for 4 h. Foam cell formation and differentiation were analysed by flow cytometry (defined as viability dyelow F4/80high Dil-positive macrophages and their mean fluorescent intensity, MFI).

BODIPY-cholesterol uptake

Murine BMDMs were incubated for 90 min with 0.025 mM TopFluor Cholesterol (BODIPY-cholesterol, Avanti Polar Lipids, Alabaster, USA) in cell culture medium. The medium was removed, and the cells were incubated overnight with 2 μg/ml acyl-coenzyme A:cholesterol O-acyltransferase (ACAT) inhibitor (Tocris Bioscience, Wiesbaden, Germany) in cell culture medium with 2 g/L bovine serum albumin (BSA). Cholesterol efflux was initiated by adding 25 μg/ml human high-density lipoprotein (HDL, Kalen Biomedical, Montgomery Village, MD, USA) to the culture media for 4 h.

The supernatants were collected, centrifuged, and analyzed with a SpectraMax M2 plate reader (Molecular Devices, San Jose, CA, USA) with a 490 nm excitation filter, 520 nm emission filter, and 515 nm cutoff. The amount of cholesterol efflux was quantified using linear regression (5-point calibration on concentrations ranging from 1 to 25 μM TopFluor Cholesterol).

BMDM proliferation assay

After differentiation, murine NOD-like receptor family pyrin domain containing 3 knockout (Nlrp3−/−) and wild-type (WT) BMDMs were incubated for 12 h in cell culture medium with 20 µg/ml human Dil-labeled oxLDL (DiI-oxLDL; Kalen Biomedical, Montgomery Village, MD, USA). In order to compare proliferation between the two different BMDM populations, 10 µmol/ml EdU was added to each well and its incorporation in proliferating cells was measured by flow cytometry (defined as viability dye negative, F4/80high CD11b-positive macrophages).

RNA Sequencing

Cells were lysed, and RNA was prepared using the RNeasy Micro Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocols. An RNA library was prepared according to the NEBNext Low Input RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA).

Paired-end sequencing was performed via the European Molecular Biology Laboratory (EMBL). Quality Control and Data Prepocessing was performed in Galaxy [38]. Tools were used with default settings unless stated otherwise. FastQC-reports were used to assess data quality repetitively during quality control and data preprocessing [1]. Cutadapt was used to trim adapters and low-quality bases [25]. Trimmed reads were mapped to a built-in reference genome (mm10) using RNA STAR [6].

After mapping, gene expression was measured using the featureCounts program and a built-in reference genome was used for annotation [14]. Raw read counts were acquired for differential expression analysis. Batch effects included the date of harvest and the origin of cells regarding individual test animals, and these were included in subsequent analysis. Differential expression analysis was performed in RStudio (RStudio-2023.06.1–524) using the packages DESeq2 and dplyr. Genes with a cumulative read count lower than 10 were filtered out. Benjamini–Hochberg correction was used for multiple testing corrections. Gene ontology analysis was performed in Metascape [43] and DAVID. Gene set enrichment analysis was performed in GSEA on the curated M2cp gene sets of the Molecular Signatures Database (MSigDB), including various gene sets that are expert-confirmed canonical representations of biological processes [34].

Aortic cell isolation and flow cytometry

Murine aortic cells were retrieved by enzymatic digestion with a two-stage procedure. First, tissue was digested with collagenase I, collagenase XI, hyaluronidase, and DNAse I solution (Sigma-Aldrich, St. Louis, MO, USA) in a thermocycler for 70 min at 250 rpm and 37 °C, followed by a second digest with collagenase II (Worthington Biochemical Corp., Lakewood, NJ, USA) and hyaluronidase for 8 min at 250 rpm and 37 °C. Murine blood samples were lysed in red blood cell lysis buffer (Biolegend, San Diego, CA, USA). Isolated cells from blood and aorta were counted using a Neubauer chamber (Marienfeld, Lauda-Königshofen, Germany). Cells were stained with specific fluorescent antibodies as specified in Supplemental Table S1.

Chimerism shifts were quantified by comparing aortic monocytes and macrophages to their circulatory precursors in blood using flow cytometry. The baseline WT/KO ratio was determined from Ly6Chigh blood monocytes (CD45.1+ or CD45.2+, CD11b+, Lin− (where Lin indicates CD3, CD19, NK1.1, Ly6G), Ly6Chigh, CD115 + , F4/80low). Aortic monocytes (Ly6Chigh, (CD45.1+ or CD45.2+, CD11b+, F4/80low or CD68+) and macrophages (CD45.1+ or CD45.2+, CD11b+, F4/80high or CD68+) were similarly analyzed. Shifts from baseline were calculated as Δ of mean WT/KO ratio in blood monocytes minus respective WT/KO ratio in aortic monocytes or macrophages. Positive Δ indicates enrichment of the respective WT population; a negative Δ indicates enrichment of KO population.

To assess proliferation and apoptosis, intracellular staining with anti-Ki-67 and anti-active Caspase-3 (Casp3) was performed using BD Cytoxfix/Cytoperm (#554,722, BD Biosciences, San Diego, CA, USA), BD Perm/Wash (#554,723, BD Biosciences, San Diego, CA, USA) and BD Permeabilization Buffer Plus (#561,651, BD Biosciences, San Diego, CA, USA), following the manufacturer’s instructions.

The gating strategy of Low-density lipoprotein receptor knockout [Ldlr−/−] chimera is shown in Supplemental Fig. S1b. Flow cytometry analysis of Apoe−/− mice was performed using the same gating strategy, as shown in Supplemental Fig. S2a.

Data were collected on a BD FACS Canto II (BD Biosciences, San Diego, CA, USA) and analyzed with FlowJo (Tree Star, Ashland, OR, USA).

Histology

Murine aortic roots were embedded in Tissue Tek Optimal cutting temperature (OCT) compound (Sakura Finetek, Tokyo, Japan) and cut into serial 5 µm cryostat sections, starting at the level of the aortic valve. Sections were stained with anti-CD68 (clone FA-11, BioRad AbD Serotec, Puchheim, Germany), anti-CD45 (AF114, R&D Systems, Minneapolis, MN, USA), secondary antibodies Donkey α-Goat AF488 (Abcam, Cambridge, UK), rabbit α-rat AF647 (Abcam, Cambridge, UK), and Click-iT EdU Imaging Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturers' instructions. Images were recorded with the Axio Imager.Z2 (Carl Zeiss Micro Imaging GmbH, Göttingen, Germany). Images were analyzed with Image Pro Premiere 9.2 (Media Cybernetics, Rockville, MD, USA) or Zeiss Zen Lite (Carl Zeiss MicroI maging GmbH, Göttingen, Germany) and manually counted. Human plaque histology methods are described in 2.2.

Statistics

Results are presented as mean ± SEM. Differences between two groups were analyzed with paired Student’s t-test as indicated in the figure legends. To assess differences between more than two groups, one-way ANOVA with Holm–Šídák test for multiple comparisons was used. p values ≤ 0.05 indicate significant changes. Pearson’s correlation coefficient was used to test for correlation.