Activated cholesterol metabolism is integral for innate macrophage responses by amplifying Myd88 signaling

Recent studies have shown that cellular metabolism is tightly linked to the regulation of immune cells. Here, we show that activation of cholesterol metabolism, involving cholesterol uptake, synthesis, and autophagy/lipophagy, is integral to innate immune responses in macrophages. In particular, cholesterol accumulation within endosomes and lysosomes is a hallmark of the cellular cholesterol dynamics elicited by Toll-like receptor 4 activation and is required for amplification of myeloid differentiation primary response 88 (Myd88) signaling. Mechanistically, Myd88 binds cholesterol via its CLR recognition/interaction amino acid consensus domain, which promotes the protein’s self-oligomerization. Moreover, a novel supramolecular compound, polyrotaxane (PRX), inhibited Myd88‑dependent inflammatory macrophage activation by decreasing endolysosomal cholesterol via promotion of cholesterol trafficking and efflux. PRX activated liver X receptor, which led to upregulation of ATP binding cassette transporter A1, thereby promoting cholesterol efflux. PRX also inhibited atherogenesis in Ldlr–/– mice. In humans, cholesterol levels in circulating monocytes correlated positively with the severity of atherosclerosis. These findings demonstrate that dynamic changes in cholesterol metabolism are mechanistically linked to Myd88‑dependent inflammatory programs in macrophages and support the notion that cellular cholesterol metabolism is integral to innate activation of macrophages and is a potential therapeutic and diagnostic target for inflammatory diseases.

d7-cholesterol, and other sterols were set as described previously (2). The measurement results were normalized by the amount of protein contained in the cell or organelle fraction, which was determined with BCA assays.

Cholesterol efflux assay
RAW cells were cultured for 24 h in DMEM containing with 0.5 μCi/ml of [ 3 H]cholesterol (PerkinElmer, Waltham, MA) and 1% FBS. After washing the cells twice with serum free medium, DMEM supplemented with 1% mouse serum, as a cholesterol acceptor, was added. Then, PRX (2 mM) and/or PSC-833 (40 µM) were added to the medium, and the cells were cultured for 20 h prior to 4-h LPS stimulation (100 ng/ml).
The chase medium was collected and subjected to brief centrifugation to pellet any residual debris. The cleared medium was used to determine the radioactive counts released from the cells. Radioactivity within the cells was determined by extraction in hexane:isopropanol (3:2) followed by solvent evaporation in a scintillation vial prior to counting.
Cholesterol efflux assays using fluorescently labeled cholesterol were carried out using a Cholesterol Efflux Assay kit (Abcam, ab196985) according to the manufacturer's protocol. Briefly, RAW cells were labelled with fluorescently labelled cholesterol for 1 h, followed by culture for 16 h in equilibration medium. The culture medium was then replaced with fresh medium supplemented with 1% mouse serum, and the cells were cultured with or without PRX (2 mM) for 20 hours prior to LPS (100 ng/ml, 4 h) stimulation. Cholesterol efflux was calculated by dividing the fluorescence intensity in the media by the sum of the fluorescence intensities in the media and cells. (The percent cholesterol efflux was calculated as 100 × (medium dpm) / (medium dpm + cell dpm)).

RNA isolation and qPCR
Total RNA was using NucleoSpin (Macherey Nagel) according to the manufacturer's protocols. StepOnePlus real-time PCR system (Applied Biosystems) was used for qPCR.
Target gene expression was normalized to the level of Gapdh for RAW cells, BMDMs, and human monocytes or the level of 18s rRNA for mouse aorta.

RNA-sequencing
Poly-A mRNA was extracted from total RNA using a NEBnext poly(A) mRNA magnetic isolation module (New England Biolab), after which RNA-seq libraries were prepared using a NEBNext Ultra RNA Library Prep kit for Illumina according to the manufacturer's protocol (New England Biolab). Libraries were PCR-amplified for ~12 cycles and sequenced on a Hi-seq 1500 (Illumina). Reads were aligned to the mm9 mouse genome using STAR (4). Expression analysis of the RNA-seq data was performed using HOMER (5) and DESeq2 (6). Gene set enrichment analyses were performed using GSEA (7) with rank files generated as previously described (http://genomespot.blogspot.com/2015/01/how-to-generate-rank-file-from-gene.html) from expression data analyzed using DESeq2. All RNA-seq data are available in the GEO under accession number GSE146236.
Sequencing data were mapped to the mm10 mouse genome. Peak calling and annotation were performed using HOMER (5). Peaks that overlapped with blacklisted regions and simple repeat regions were removed. HOMER-identified peaks whose scores were ≥30 were selected as high-confidence peaks and used to identify the likely target genes using GREAT (8) with the default parameters.
For identification of LXR target genes, published LXR ChIP-seq and corresponding input data (GSM2095083, GSM2095084, GSM2095087, GSM2095163, GSM2095164) were mapped to the mm10. HOMER (5)-identified peaks whose scores were ≥20 were selected and used to identify target genes using GREAT (8). To further identify genes positively regulated by LXR, RNA-seq data (GSM2095128, GSM2095130, GSM2095179, GSM2095180) were mapped to the mm10. Genes upregulated by the LXR agonist GW3965 were identified using DESeq2 (6). Genes related to LXR peaks and upregulated by GW3965 were used as the LXR target genes for the analysis in Fig. 6.

Plasmids
Full length cDNAs for mouse wild-type Myd88 and its CRAC mutants were cloned into pCMV-Flag, pCAGGS-CFP (a generous gift of Dr. T. Taniguchi, University of Tokyo), pGEX-4T-1 (GE Healthcare) vectors to obtain Flag-or CFP-tagged proteins and Myd88-GST fusion protein.

Luciferase reporter analysis
Using Fugene6 (Roche), HEK293T cells were transiently transfected with luciferase reporter plasmids (pNF-kB-Luc and pRenilla-Luc) together with a series of mouse Myd88 expression plasmids. Luciferase activity was measured using a dual-luciferase reporter assay system (Promega).

Cholesterol binding assay
To assess cholesterol binding to Myd88, 1 µg of GST-Myd88 or untagged GST was pre- NBD-cholesterol was excited at 490 nm, and the fluorescence emission spectra were recorded in the range of 510-600 nm. The dissociation constant (Kd) was calculated as described previously (11).

Mouse IL-6 ELISA
Culture medium was collected from RAW cells and analyzed using a mouse IL-6 ELISA kit (R&D) according to the manufacturer's instructions (n=6). For immunohistochemical analysis, isolated aortic trees and roots were fixed in 4% paraformaldehyde in PBS and then frozen and cut into 6-μm sections. To quantify areas of atherosclerotic lesions, fixed aortic trees and roots were stained with oil red O in 60% isopropanol. Oil red O-positive areas were quantified using ImageJ.

Atherosclerosis study in mice
Immunohistochemical staining was performed as previously described (13). Briefly, cryosections were fixed in 4% paraformaldehyde/PBS for 10 min at room temperature.
The sections were then blocked in 5% BSA/PBS blocking solution, after which primary antibodies prepared in blocking solution were added and incubated overnight at 4°C.
After three washes in PBS, the sections were counterstained with Hoechst. Histological images were captured using an Olympus IX73 microscope.

Human atherosclerosis study
To quantify atherosclerotic states in humans, IMT in the common carotid arteries was was considered an integral indicator of mean IMT.

Human monocyte study
After collecting whole blood from healthy donors, monocytes were isolated by centrifuging the blood on a Ficol density gradient followed by magnetic CD14-positive separation using MACS CD14-positive microbeads (Miltenyi Biotec) and MACS separation columns (Miltenyi Biotec). The isolated monocytes were then seeded into sterile 24-well culture plates at a density of 10 6 cells/well and incubated in RPMI containing 10% FBS without colony-stimulating factors. The cells were cultured for 7 days at 37°C in a humidified CO2-incubator (95% air and 5% CO2). On day 7, the medium was replaced with serum-free X-VIVO medium for 1 day, after which 2 mM PRX was added. After an additional 24 h, LPS was added to a final concentration of 100 ng/ml.

siRNA-mediated knockdown
For siRNA-mediated gene knockdown, chemically synthesized siRNA targeting genes of interest and control siRNA (siPerfect Negative Control) were purchased from Sigma.
RAW cells were transfected with 50 nM siRNA using Lipofectamine RNAiMAX reagent (Thermo) according to the manufacturer's protocol. Forty-eight hours after transfection, the cells were used for experimentation.

TLR3, TLR9, and RIG-I activation
RAW cells were stimulated with poly(I:C), CpGB or 3pRNA (at a final concentration of

High-performance liquid chromatography
For lipoprotein (CM, VLDL, LDL and HDL) distribution analyses, plasma samples from 4 or 5 mice per group were analyzed using an upgraded high-performance liquid chromatography (HPLC) technique as previously described (Skylight Biotech) (14).

Statistical analysis
Sample sizes were not based on power calculations. No animals were excluded from analyses. Comparisons between two groups were made using two-tailed Student's t tests.
Differences among more than two groups were analyzed using one-way ANOVA