2. Materials and Methods

2.1. Materials
RPMI 1640, fetal bovine serum, streptomycin sulfate, penicillin G sodium and amphotericin B were purchased from Gibco BRL (Gaithersburg, MD, USA). Mineral oil (Nujol) was purchased from Schering-Plough (Sao Paulo, Brazil). Phenyl methyl sulfonyl fluoride (PMSF) was purchased from Sigma-Aldrich (St Louis, MO, USA). A Protein G column and a PD-10 desalting column were purchased from GE Healthcare (Uppsala, Sweden). All synthetic peptides, including FITC-labeled peptides, were synthesized and purified by HPLC to purities greater than 95% by the Chinese Peptides Company (Hangzhou, China). The 1A3H2 and 2C7D5F10 anti-LDL (−) mAbs were obtained as previously described [11].

2.2. Phage Display Random Peptide Libraries
Two phage display random peptide libraries were previously constructed on vector fUSE55 displaying inserts X6 or CX8C (C indicates cysteine; X indicates any amino acid) that were fused to an outer minor phage coat protein (pIII) at the N-terminus of an Fd phage [24,25]. These tetracycline-resistant libraries consisted of a linear, 6-peptide configuration (X6), or a cyclic, 8-peptide configuration (CX8C) with a loop scaffold developed by flanking the random peptide sequence with a pair of cysteine residues to form a bridge. The details of biopanning, binding assays, and ELISA procedures with synthetic peptides are described in the Supplementary Materials (A. Phage Display).

2.3. Characterization of the Peptides by Small-Angle X-ray Scattering (SAXS)
SAXS experiments were performed with SAXS Xenocs-XeussTM (Xenocs, Sassenage, France) with a GENIX source and Fox2D mirrors. Collimation was performed using two sets of scatterless slits that provide a high flux (approximately 0.5 × 108 ph/s/mm2) with very low parasitic scattering. Data treatment was performed using the software package SUPERSAXS, and the final scattering intensity was normalized to an absolute scale based on water as the primary standard. The normalization of the data to an absolute scale permitted the estimation of the average molecular weight of the particles [26]. The methodological details are described in the Supplementary Materials (B) SAX Structure Characterization.

2.4. Isolation of Human LDL (−)
Venous blood from normocholesterolemic volunteers was collected into tubes containing ethylenediamine tetraacetic acid (EDTA, 1 mg/mL blood). Pooled plasma was immediately separated by centrifugation, at 2500 rpm for 10 min at 4 °C and 1 mM (PMSF); 2 mM benzamidine, 2 μg/mL aprotinine and 20 mM butylated hydroxytoluene (BHT) were added to prevent protease activity and oxidative reactions. LDL (d ~ 1.019 to 1.063 g/mL) was isolated from plasma by preparative sequential ultracentrifugation, using an Optima XE-90 ultracentrifuge (Beckman Coulter, IN, USA) with a fixed angle rotor (T 70.1). Plasma was centrifuged with saline (density = 1.019 g/mL) at 56,000 G for 7 h at 4 °C and the top fraction was removed. The remaining plasma was adjusted with KBr to a density of 1.063 g/mL and centrifuged with saline (density = 1.063 g/mL). The top LDL fraction was removed and dialyzed against 20 mM Tris-HCl buffer, pH 7.4, 20 mM BHT, and 10 mM EDTA for 4 h, at 4 °C, protected from light, with buffer exchanges each 1 h. The LDL (−) was separated from the native LDL (nLDL) by fast protein liquid chromatography (FPLC, ÄKTA Start, GE Healthcare, Chicago, Illinois, USA) using an ion exchange column (Sepharose UNO Q, Bio-Rad Laboratories Inc, Hercules, CA, USA). Total LDL was injected into the column pre-equilibrated with buffer A (10 mM Tris-HCl, pH 7.4) and eluted with a multi-step gradient of buffer B (10 mM Tris-HCl, 1 M NaCl). The isolated LDL (−) fraction was dialyzed against phosphate buffered saline (PBS) and concentrated using a specific device (Vivaspin 20, 100,000 MWCO, GE Healthcare Life Sciences, Uppsala, Sweden). The presence of LPS in LDL samples isolated from human plasma was determined by the limulus amebocyte lysate (LAL) test (Lonza, Verviers, Belgium). Supplies used for LDL isolation and cell culture were treated with E-Toxa-Clean ®Concentrate (Sigma Aldrich, St. Louis, MO, USA). The endotoxin levels of LDL (−), P1A3, and P2C7 peptides were < 0.1 EU (endotoxin unit) for all samples.

2.5. Obtention of Bone Marrow-Derived Macrophages
Bone marrow-derived macrophages (BMDMs) were obtained by extracting bone marrow cells from femurs of C57BL/6 mice according to [27]. Further, bone marrow cells were differentiated to macrophages through incubation with RPMI 1640 media, with 10 ng/mL of recombinant M-CSF (Peprotech, Rocky Hill, NJ, USA) and 15% fetal bovine serum at 37 °C, 5% CO2 in humidified air. BMDMs were characterized using flow cytometry and an F4/80-PE-Cy5 antibody (eBioscience, San Diego, CA, USA), which is a specific antibody marker for murine macrophages. Labeled cells were analyzed in a flow cytometer (FACS Canto, BD Biosciences, San Diego, CA, USA). FACS characterization of macrophages is shown in the Supplementary Materials (C. Macrophage characterization; Figure S6).

2.6. Peptide Endocytosis by Macrophages
After differentiation, BMDMs (105 cells/well) were plated on glass-bottomed culture plates (35 mm diameter with 4 wells) (Greiner Bio-One, Kremsmunster, Austria). The cells were maintained in 1% FBS before treatment. Then, the cells were incubated with 100 μg/mL Cy5-P1A3 and Cy5-P2C7 for 3 and 6 h. Further, the cells were fixed in 4% paraformaldehyde before visualization under a Zeiss LSM 780-NLO confocal microscope (Carl Zeiss, Jena, Germany). In order to investigate if the internalization of peptides by macrophages occurred by endocytosis, inhibitors of this process were used as follows. For this assay, 106 cells/well were incubated for 1 h with the following inhibitors: 1 mM sodium azide (oxidative phosphorylation inhibition), 1 μM Brefeldin A (Golgi complex/endoplasmic reticulum protein traffic inhibition), 40 μM Cytochalasin D (phagocytosis inhibition), 80 μM Dynasore (dynamin-dependent endocytosis inhibition), and 20 μM sodium monensin (lysosome enzyme inhibition). The cells were washed with PBS, then 100 μg/mL Cy5-P1A3 or Cy5-P2C7 were added and the cells were incubated for up to 2 h.

2.7. Macrophage Activation
BMDMs (105 cells/well) were incubated with synthetic peptides and 100 μg/mL LDL (−) or PBS as negative control and 10 μg/mL LPS as positive control for 3 h at 37 °C. Total RNA was extracted with TRIzol (Life Technologies, Carlsbad, CA, USA) following the manufacturer’s instructions. The extracted RNA was quantified by spectrophotometry. cDNA was constructed from RNA samples by RT-PCR with Superscript Vilo (Life Technologies, Carlsbad, CA, USA). qPCR was performed using the 7500 Fast Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA) to analyze the expression levels of NOS2, COX-2, TNF-α, IL-1α, TGF-β, and IL-10. Activation of macrophages by LPS was evaluated as a positive control (Supplementary Materials (C. Macrophage characterization; Figure S7)). To verify if BMDM activation was dependent on the endocytosis of peptides an experiment was carried out with Brefeldin A. For this 106 BMDM/well were treated for 1 h with 1 μM Brefeldin A. Then, the cells were washed with PBS and incubated with 100 μg/mL P2C7 in RPMI 1640, 10% FBS and Brefeldin A (1 μM) for 3 h. A negative control was done only with RPMI 1640, 10% FBS and Brefeldin A (1 μM) incubated for 3 h. Further the expression of NOS2, TNF-α and TGF-β was evaluated by qPCR assay.

2.8. Evaluation of BMDM Polarization by Flow Cytometry
For this assay, 106 cells/well were incubated for 48 h with 100 μg/mL P2C7 or PBS as the negative control. After incubation, macrophages were washed with PBS and removed from the plate with cold PBS (plus 10 mM EDTA). The cells were stained with anti CD206—FITC (Invitrogen, MR5D3), anti-F4/80—PE-Cy5 (eBioscience, BM8), anti-CD80-PE (eBioscience, 16-10A1), anti-F4/80—FITC (BM8), anti-MHC II—APC-Cy7 (Invitrogen, M5/114.15.2) and anti-CD86—APC (Invitrogen, GL1) in PBS, 4% FBS for 30 min. Further labeled cells were analyzed in a flow cytometer (FACS Canto, BD Biosciences, San Diego, CA, USA). FACS characterization of M2 macrophages is shown in the Supplementary Materials (C. Macrophage characterization; Figure S8).

2.9. Cytokine Quantification by Cytometric Bead Array (CBA) Kit
BMDMs (106 cells/well) were incubated with synthetic peptides at 100 μg/mL for 24 h in an incubator at 37 °C. Then, the medium was collected and stored at −80 °C. Cytometric bead array (CBA) mouse inflammation kits (BD Biosciences, San Diego, CA, USA) were used according to manufacturer instructions to quantify the levels of IL-12p70, TNF-α, INF-γ, CCL2, IL-10, and IL-6 cytokines.

2.10. Nitric Oxide Quantification (NOA)
The NO concentration in the culture medium was evaluated by measuring the NO2− accumulation with a nitric oxide analyzer (NOATM 280; Sievers Inc., Boulder, CO, USA) that is based on a gas-phase chemiluminescence reaction between NO and ozone. In brief, BMDMs (2 × 106 cells) were treated for 24 h with 100 μg/mL LDL (−), P1A3 or P2C7, and 100 μL of culture supernatants were injected into a reflux chamber containing vanadium (III) in 3N HCl heated to 90 °C. The NO produced was detected by gas phase chemiluminescence after reaction with ozone. A calibration curve was then created using a sodium nitrate standard solution.

2.11. Statistical Analysis
Statistical analyses were performed using GraphPad Prism software (version 6.0, San Diego, CA, USA) and one-way ANOVA followed by Tukey–Kramer or Dunnett’s tests for multiple comparisons as well as t-tests for comparisons between data pairs.

2.12. Data Availability
The data sets that were generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.

2.13. Human and Animal Rights
The study was approved by the Committee on Ethics in Human Research (CEP) for the use of human blood (protocol number 557), and by the Committee on Ethics in Animal research (CEUA) for the use of C57BL/6J (protocol number 492), both of the Faculty of Pharmaceutical Sciences from the University of Sao Paulo, in agreement with the National Committee on Ethics in Research (CONEP) and the Brazilian College for Animal Experimentation (CONCEA) guidelines. Informed consent was obtained from all study subjects.