Increased LL37 in psoriasis and other inflammatory disorders promotes LDL uptake and atherosclerosis

Patients with chronic inflammatory disorders such as psoriasis have an increased risk of cardiovascular disease and elevated levels of LL37, a cathelicidin host defense peptide that has both antimicrobial and proinflammatory properties. To explore whether LL37 could contribute to the risk of heart disease, we examined its effects on lipoprotein metabolism and show that LL37 enhanced LDL uptake in macrophages through the LDL receptor (LDLR), scavenger receptor class B member 1 (SR-B1), and CD36. This interaction led to increased cytosolic cholesterol in macrophages and changes in expression of lipid metabolism genes consistent with increased cholesterol uptake. Structure-function analysis and synchrotron small-angle x-ray scattering showed structural determinants of the LL37-LDL complex that underlie its ability to bind its receptors and promote uptake. This function of LDL uptake is unique to cathelicidins from humans and some primates and was not observed with cathelicidins from mice or rabbits. Notably, Apoe–/– mice expressing LL37 developed larger atheroma plaques than did control mice, and a positive correlation between plasma LL37 and oxidized phospholipid on apolipoprotein B (OxPL-apoB) levels was observed in individuals with cardiovascular disease. These findings provide evidence that LDL uptake can be increased via interaction with LL37 and may explain the increased risk of cardiovascular disease associated with chronic inflammatory disorders.

fluorescence analysis (n=4 per each group) (F) are shown.Scale indicates 20 μm (E).(G) Quantitative fluorescence analysis of THP-1 cells treated with pHrodo-LDL and LL37 in the presence of PC (n=3 per each group).(H, I) Biotinylated-LDL was cultured with LL37 in the presence of PC.The samples were subjected to co-immunoprecipitation with anti-biotin antibody, and the co-immunoprecipitation samples were then immunoblotted with anti-LL37 antibody.
Representative immunoblotting image (H) and quantitative analysis of LL37 signal intensity

Flow cytometric analysis
For evaluation of LDL uptake in vitro, THP-1 cells were treated with 1.25 μg/ml of pHrodo-LDL and LL37.Eighteen hours after that, cells were isolated by pipetting.For evaluation of LDL uptake in vivo, 1ml of 4% thioglycolate (VWR) was injected into mouse peritoneal cavity.Forty-eight hours after that, 1.25 μg/ml of pHrodo-LDL in 100uL of PBS was injected intraperitoneally.
Eighteen hours after the injection of pHrodo-LDL, peritoneal cells were isolated.The isolated cells (THP-1 or mouse peritoneal cells) were incubated in a FACS staining buffer (PBS containing 5% BSA and 0.01% NaN3) with human Fc receptor binding inhibitor polyclonal antibody (eBioscience) or mouse CD16/32 antibody (eBioscience).Then, cells were stained with antibodies.
LDL particles were incubated with LL37, LL34, and Cramp at a peptide-to-lipid (P/L) molar ratio of 3/35.SAXS experiments were conducted at Stanford Synchrotron Radiation Lightsource (SSRL, Beamline 4-2) using monochromatic X-rays of wavelength λ =1.378 Å (energy 9keV).A Pilatus3 X 1M detector (pixel size 172 μm) was used to collect the scattering signal.Multiple measurements were performed on each sample to ensure data quality.The two-dimensional (2D) powder diffraction pattern was integrated with the Nika package 1.81 in Igor Pro 9.The integrated intensity I(q) was plotted against the q, where q was the magnitude of the scattering vector defined as q=(4πsinθ)/λ with θ the scattering angle and λ the wavelength of the X-rays.The form factor fitting was done using the SasView 5.0.6 (the core-shell ellipsoidal model) and the best fit in the lower range of q was obtained by adjusting the radius of the shell (r), the shell thickness (t), the ratio of the shell thickness at the pole to the equator (λ), the aspect ratio (ξ), and the relative electron density of the core with respect to the shell (ρ).

qRT-PCR
The RNA was isolated from the THP-1 cells or HMDMs using Purelink RNA isolation columns (ThermoFisher Scientific) according to the manufacturer's instructions.RNA was quantified using a Nanodrop spectrophotometer (ThermoFisher Scientific), and up to 1000 ng of RNA was reversetranscribed using Verso cDNA Synthesis Kit (ThermoFisher Scientific).Quantitative real-time PCR reactions were run on a CFX96 real-time detection system (Bio-Rad) using gene-specific primers and SYBR green master mix (Biomiga Inc).PCR primers were synthesized by Integrated DNA

Isolation of peritoneal macrophages
Peritoneal lavage was harvested from wild type mice.Collected peritoneal cells were resuspended in RPMI medium, and cultured for 2 hours at 37 °C.Nonadherent cells were removed by gently washing by PBS, and adherent cells were used as peritoneal resident macrophages for analyses.

Evaluation of atherosclerotic plaques and lipid analyses
Mice were anesthetized with isoflurane, blood was collected for lipid analyses, and the vascular system was perfused with PBS followed by 4% paraformaldehyde by left ventricle puncture.The heart and whole aorta were isolated and placed in 4% paraformaldehyde for fixation overnight.The heart was embedded in OCT compound, and frozen sections (8 μm) of the aortic sinus were obtained.The sections of the aortic sinus were stained with freshly prepared Oil red O working solution for 20 minutes.Then, the sections were rinsed with PBS and counterstained with Mayer's hematoxylin.For en face analysis, the thoracic aorta was cleaned.Then, the aorta was opened longitudinally and pinned, and was stained with the Oil Red O working solution.Plaque areas and relative to heavy chain (n=3 per each group) (I) are shown.Error bars indicate mean ± SEM; ***p<0.001using Student's t test.N.S: not significant, SAXS: small angle X-ray scattering, PC: phosphatidylcholine of LL37 (n=4 per each group).(E) PLA on HMDMs for LL37 associated with LDLR, SR-B1 or CD36 ± LDL (n=4 per each group).(F) PLA on THP-1 cells for biotinylated LDL association with LDLR, SR-B1 or CD36 after addition of Cramp (n=4 per each group).(G) PLA on THP-1 cells for Cramp associated with LDLR, SR-B1 or CD36 ± LDL (n=4 per each group).(H, I) Dil-LDL associated with THP-1 (H) or HMDMs (I) after addition of LL37 or Cramp (n=4 per each group).Error bars indicate mean ± SEM; *p<0.05,**p<0.01,***p<0.001,****p<0.001using Student's t test (A-C) or one-way ANOVA multiple-comparison test (D-I).HMDMs: human monocyte-derived macrophages Supplemental Figure 3. LL37 and LDL increases intracellular lipid and alters macrophage gene expression.(A-C) Representative images of HMDMs treated with LDL ± LL37 after staining with filipin (blue) to detect free cholesterol (A), or with Nile red (red) to detect lipid and with DAPI (blue) to detect DNA (B), or with Bodipy (green) to detect lipids and DAPI (blue) to detect DNA (C).Scale indicates 50 μm (A) or 20 μm (B, C). (D) Quantitative analysis of signal intensity in HMDMs after Bodipy staining as in (C) (n=3 or 4 per each group).(E) qPCR quantification of mRNA expression for indicated genes in HMDMs treated with LDL ± LL37 (n=4 per each group).Error bars indicate mean ± SEM; *p<0.05,**p<0.01,***p<0.001,****p<0.001using one-way ANOVA multiple-comparison test.HMDMs: human monocyte-derived macrophages Supplemental Figure 4. Transgenic expression of CAMP enhances development of atherosclerosis.(A) Weight change of Apoe -/-mice and LL37 tg/tg Apoe -/-mice fed normal or high fat diet (n=4 and 5 in Apoe -/-mice and LL37 tg/tg Apoe -/-mice with normal diet, respectively.n=8 and 10 in Apoe -/-mice and LL37 tg/tg Apoe -/-mice with high fat diet, respectively).(B) Serum concentration of HDL cholesterol and triglyceride in Apoe -/-mice and LL37 tg/tg Apoe -/-mice fed normal or high fat diet for 10 weeks (n=4 per each group with normal diet, n=8 per each group with high fat diet).(C) Size-exclusion FPLC (fast protein liquid chromatography) analysis of serum from LL37 tg/tg Apoe -/- mice with high fat diet to determine cholesterol and triglycerides in each fraction (n=1 per each fraction).(D) LL37/ hCAP18 concentration measured by ELISA in each fraction of the FPLC (n=1 per each fraction).(E) Immunoblot analyses of indicated fraction of the FPLC with antiapolipoprotein B (apoB) antibody and anti-LL37 antibody.(F) Proportion of LL37/ CD68 stained areas within whole LL37 stained areas in the plaque of LL37 tg/tg Apoe -/-mice fed high fat diet (n=10).Error bars indicate mean ± SEM; N.S: not significant.ND: normal diet, HFD: high fat diet

Table 2 . Sequences and catalog number of PCR primers
After that, cells were immediately imaged to prevent photo bleaching.For Nile red staining, fixed cells were stained with Nile red (Thermo Fisher Scientific) for 1 hour at room temperature.For Bodipy staining, fixed cells were incubated with Bodipy 493/ 503 (1: 1000 dilution, Thermo Fisher Scientific) for 30 minutes.Nuclei were counterstained with 4′,6-diamidino- 2-phenylindole (DAPI).All images were taken with an Olympus BX41 microscope.For quantification of Bodipy staining, 18 images were collected for each condition, and Bodipy/ DAPI signal was calculated by Image J.LDL aggregate analysisDil-LDL (1.25 μg/ml) was incubated with indicated peptides in PBS for 18 hours.Aggregate fluorescence was evaluated using the microscope.After LDL aggregate was washed by PBS, the aggregate was lysed by 1% SDS and Dil-signal was also quantified on Spectramax microplate reader (Molecular Biosystems) with at excitation 530 nm/ emission 580 nm excitation.
Technologies and the sequences or catalog number are shown on Supplemental Table2.