The protocol of the clinical study has been described in detail in the previous publication (Rysa et al., 2013). In short, healthy volunteers, aged 18–40 years, were recruited for the study. The study had a randomized, open, and placebo-controlled crossover design. Twelve individuals were administered 600 mg rifampicin (Rimapen; Orion Corporation, Espoo, Finland), an efficient PXR agonist, or placebo daily for a week with at least a 4-week washout period before the next treatment arm. At the end of each arm, blood samples were collected on the morning of the eighth day. The study was approved by the ethics committee of the Northern Ostrobothnia Hospital District (Oulu, Finland) and the Finnish Medicines Agency. Written, informed consent was obtained from each subject. The study procedures performed were in accordance with the ethical standards of the Helsinki Declaration. This trial was registered at ClinicalTrials.gov as NCT00985270.

Total cholesterol, low-density lipoprotein cholesterol (LDL), high-density lipoprotein cholesterol (HDL), and triglycerides were assayed with an automatic chemical analyzer (Advia 1800; Siemens Healthcare Diagnostics GmbH, Eschborn, Germany) at the clinical laboratory of Oulu University Hospital (NordLab, Oulu, Finland) with a method validated for clinical use.

The analysis of 4βHC, 25HC, and 27HC was done at VTT Technical Research Centre of Finland Ltd. (Espoo, Finland) as described in (Hukkanen et al., 2015). Briefly, a 30 µl aliquot of serum was extracted with 400 µl chloroform:methanol (2:5, v:v) mixture, after addition of 10 µl of internal standard mixture (4βHC-D7, 25HCD6, desmosterol-D6, and 4(R/S),25-epoxycholesterol-D6; c = 0.25 mg/L) and after evaporation, the sample was derivatized with 40 µl of N-Methyl-N-(trimethylsilyl)trifluoroacetamide. The GC–MS system consisted of an Agilent 6890 gas chromatograph (Agilent Technologies, Santa Clara, CA, USA) combined with Agilent 5973 mass selective detector (MSD). Selective ion monitoring, using specific masses for each target analyte, was used in the detection. The calibration curves were constructed on the range of 1–200 ng/ml. The concentration of 25HC was below the limit of quantification in two subjects (one subject during rifampicin arm, another subject during placebo arm). In these cases, 25HC concentration was set as the half of the limit of quantification for statistical analyses.

Liquid chromatography–electrospray–high-resolution mass spectrometry was utilized for the measurement of 4βHC in rat serum at the Admescope Ltd. (Oulu, Finland)(Hautajarvi et al., 2018). The concentration of 4α-hydroxycholesterol was measured simultaneously to control for the sample storage conditions.

Blood samples from the study subjects were collected into BD Vacutainer^® CPT Cell Preparation tubes with sodium citrate (BD, Franklin Lakes, NJ). After 30 min incubation at room temperature (RT), CPT tubes were centrifuged at 1800 × g for 30 min at 22°C, and the isolated mononuclear cells were collected. They were washed (1× PBS, 2 mM EDTA), centrifuged at 800 × g for 8 min at 22°C and resuspended in red blood cell lysis buffer (155 mM NH₄Cl, 10 mM KHCO₃, 2 mM EDTA). The cells were centrifuged at 200 × g for 5 min at 22°C, and the washes with lysis buffer were repeated if necessary. The purified mononuclear cells were washed twice with washing buffer by centrifuging at 200 × g for 5 min at 22°C. The cell suspension was lysed by suspending in RLT-buffer (RNeasy^® Mini Kit; Qiagen, Hilden, Germany) containing 1%-β-mercaptoethanol (Sigma-Aldrich). The cell lysate was stored at −70°C.

For the homogenization, human mononuclear cell lysates were centrifuged at 16,000 × g for 2 min through QIAshredder (Qiagen) spin columns. From the homogenized flow through, total cellular RNA was isolated and purified using RNeasy^® Mini Kit columns and solutions (Qiagen) with DNase treatment according to the manufacturer’s instructions. The samples were stored at −70°C until use.

A 1 μg aliquot of the purified total cellular RNA from the cell samples was first converted to cDNA using Moloney-Murine Leukemia Virus (M-MuLV) reverse transcriptase and random hexamer primers of RevertAid First Strand cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA). For the qRT-PCR, the target genes were amplified in duplicates with iQ5 Multicolor Real-Time PCR Detection System iCycler using iQTM SYBR^® Green Supermix (Bio-Rad). The primers used are described in Supplementary Table 1. The results were analyzed by ΔΔCT algorithm with iQ5 Optical System Software (Bio-Rad). The relative gene expressions were calculated by normalization against human glyceraldehyde-3-phosphate dehydrogenase.

The quality and integrity of the isolated human in vivo mononuclear cell RNA from the study volunteers (n = 12) was monitored by QIAxcel RNA QC Kit v2.0 (Qiagen) according to the manufacturer’s instructions. All the RNA Integrity Score values were above 7.4, indicating high-quality RNA with little degradation. RNA samples where first labeled using the Illumina Total PrepT RNA Amplification Kit (Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions, with 350 ng of RNA per sample. The quality of all cRNA samples was controlled on 2100 Bioanalyzer RNA nano chips (Agilent Technologies) and quantified using a Nanodrop 2000 spectrometer (Thermo Fisher). A total of 750 ng of each sample was used in the Direct Hybridization Assay Workflow (Illumina, San Diego, CA) using HumanHT12_V4 expression beadchips (Illumina). The beadarrays were scanned using a HiScan instrument (Illumina). The work was carried out at the Core Facility of the Estonian Genome Center, University of Tartu, Estonia. Probe intensity and detection data were obtained using Illumina BeadStudio software, and further processed with GeneSpring GX 12.6 software (Agilent Technologies). The raw expression data was quality assessed, log2-transformed, and subjected to background adjustment and normalization. Genes were defined as differentially expressed if the fold change was at least 1.2-fold as compared to respective controls and statistically significant (P < 0.05, paired t-test and Benjamini and Hochberg false discovery rate). The complete data sets are available from the NCBI’s Gene Expression Omnibus (GEO) database (accession number GSE108100), and gene expression profiling data comply with the MIAME standard.

Blood from one healthy female volunteer was collected into CPT tubes, and mononuclear cells were isolated as described above. The mononuclear cell suspension was centrifuged at 200 × g for 5 min at 22°C followed by suspension of the isolated cells in growth medium (1%-penicillin–streptomycin in DMEM, Sigma-Aldrich). The cells were plated and after 2-h adheration washed with PBS. Serum-free Macrophage-SFM medium (Invitrogen, Carlsbad, CA), 5 ng/ml Granulocyte-Macrophage-Colony-Stimulating-Factor (GM-CSF) and 1% penicillin–streptomycin was added to initiate the differentiation of monocytes into macrophages at 37°C, 5% CO₂. The macrophage medium was changed every 2–3 days, and after 7 days the differentiation was confirmed by the phenotype analysis and by immunofluorescence staining with a mature-macrophage specific antibody MCA1122 (Bio-Rad, Raleigh, NC). For the qRT-PCR experiments, the macrophages were incubated for 18 h in growth medium without GM-CSF with 4βHC (1 µM, 10 µM, and 20 µM), 22R-hydroxycholesterol (22RHC; 10 µM and 20 µM, a positive control), rifampicin (10 µM and 20 µM), and ethanol (vehicle control for hydroxycholesterols) and dimethyl sulfoxide (vehicle control for rifampicin). The qRT-PCR was performed as described above.

Foam cells were generated by incubating macrophages (unexposed to study compounds) in LDL pool solution. The LDL pool was prepared by ultracentrifugation from human donor blood and acetylated with acetic anhydride (AcLDL pool). The differentiated macrophages cultured on 24-well-plates were washed 3 times with 1× PBS. AcLDL solution was added to a final concentration of 50 μg/ml diluted in growth medium without GM-CSF. In the rest of the wells plain growth medium without GM-CSF was added for reference control. Macrophages were incubated for 48 h at 37°C, 5% CO₂ to produce foam cells. The phenotypic change was observed by light microscope and Oil Red O staining. The foam cells were incubated with the study compounds for 18 h, and qRT-PCR was performed as described above. The experiments on macrophages and foam cells were performed in four separate wells per experimental condition (n = 4).

Blood from one healthy female volunteer was collected, and macrophages were generated as described above. Foam cells for functional cholesterol transport experiments were generated from macrophages by incubating with [1α,2α(n)-³H]-cholesterol oleate-labeled acetylated LDL ([³H]-AcLDL). For the efflux and influx experiments, [³H]-AcLDL was added in each well to a final concentration of 50 μg/ml diluted in growth medium without GM-CSF and simultaneously exposed to a study compound or vehicle control. Macrophages were incubated for 24 h at 37°C, 5% CO2 to produce foam cells and then cells were washed 3 times with 1× PBS.

Then ApoAI and HDL₂ acceptor pools prepared in efflux medium (DMEM, Sigma-Aldrich) were added into wells to a final concentration of 10 μg/ml and 50 μg/ml, respectively, and efflux medium without acceptors was used as control. Cells were incubated for 18 h at 37°C and 5% CO₂. The mediums were collected and filtered through Nunc 96 Filter Plates (Fritted 96 DeepWell Plate, pore size 20 μm, Thermo Fisher) by centrifuging shortly at 100 × g. The cells were washed twice with 1× PBS and lysed in 0,1M NaOH solution by incubating for 2 h at RT on a plate stirrer. Liquid scintillation fluid Optiphase Supermix solution (Wallac, Perkin Elmer, Waltham, MA) was added to the mediums and cell lysates, and emissions were measured by scintillation and luminescence counter (1450 LSC & Luminescence Counter, Wallac, MicroBeta Trilux, Perkin Elmer). The protein concentrations of the cell lysates were determined according to the manufacturer’s instructions (DC Protein Assay Kit II, Bio-Rad). The influx and efflux experiments were performed in four separate wells per experimental condition (n = 4).

The cholesterol efflux (%) from foam cells was calculated using the following formula: [(counts per minute in medium)/(counts per minute in medium + counts per minute in cell lysate) x 100]. Furthermore, the efflux (%) was normalized to the cell protein concentration (%/μg). The cholesterol influx was calculated as the sum of counts per minute in medium and counts per minute in cell lysate normalized by cell protein content in each well (cpm/μg).

Male Sprague Dawley rats were dosed with 40-mg/kg pregnenolone 16α-carbonitrile (PCN) or vehicle control (corn oil plus 30% dimethyl sulfoxide) for 1, 3, or 6 days (five rats per group) as previously described (Rysa et al., 2013). The rats dosed with PCN vs. vehicle control are an established model for the study of the effects of PXR activation. The dosing was randomized; no blinding was undertaken. At the end of the experiment, the rats were decapitated, and the organs were immersed in liquid nitrogen and stored at −70°C. The experimental designs were approved by the national Animal Experiment Board in Finland. Animal studies are reported in compliance with the ARRIVE guidelines (Kilkenny et al., 2010; Mcgrath and Lilley, 2015). The investigation conforms to the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the US National Institutes of Health.

Total RNA was isolated using RNAzol reagent (Sigma-Aldrich) according to the manufacturer’s protocol. One microgram of RNA was reverse transcribed to produce complementary DNA using p(dN)6 random primers and RevertAid reverse transcriptase. The qRT-PCR reactions were performed using FastStart Universal SYBRGreen Master Mix or FastStart Universal Probe Master Mix (Roche, Basel, Switzerland). The sequences of the rat primers are presented in Supplementary Table 2. Fluorescence values of the qRT-PCR products were corrected with the fluorescence signals of the passive reference dye (ROX; Roche). The RNA levels of target genes were normalized against the 18S control levels using the ΔΔCT method.

Total protein was extracted from the left ventricle of the heart of vehicle or PCN-treated rats (5 rats per group) in 0.1 M phosphate buffer supplemented with Halt™ protease and phosphatase inhibitor cocktail. Protein concentrations were measured using the Bradford protein assay. Aliquot of 25 µg protein was separated on a 10% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane. Blots were blocked for 1 h at room temperature with 1% Milk (ECL prime™ blocking reagents; GE Healthcare, Chicago, IL, United States) in TBS buffer containing 0.05% Tween 20, then incubated overnight at +4°C with ABCA1 rabbit polyclonal antibody (NB400-105SS, 1:500; Novus Biologicals, Abingdon, UK), ABCG1 rabbit polyclonal antibody (NB400-132SS,1:1,000; Novus) and mouse monoclonal anti-β-actin antibody (A1978, 1:5,000; Sigma-Aldrich), antibodies were diluted in TBS buffer containing 5% milk and 0.05% Tween 20. Then membranes were washed 5 times 5 min with TBS buffer containing 0.1% Tween 20 before finally incubated with secondary antirabbit antibody (SC-2004, 1:1,500; Santa Cruz Biotechnology) or antimouse antibody (NA931, 1:10,000; Amersham ECL, GE Healthcare) 1 h at room temperature. The immunoreactive bands were detected by chemiluminescence (ECL start Western Blotting Detection Reagents; GE Healthcare) and quantified (Odyssey^® FC imaging system; LI-COR Biosciences, Lincoln, NE, USA). The amount of the ABCA1 and ABCG1 protein was normalized against the β-actin.

The parameters of the volunteer study were compared across treatments by paired two-tailed Student’s t-test. The intraindividual ratio (rifampicin to placebo) of the expression of gene transcripts in mononuclear cells in vivo was analyzed with one-sample t-test. Unpaired two-tailed Student’s t-test (when two groups) and ANOVA with Dunnett’s post hoc test (when more than two groups) were used to analyze the cell and animal experiments. The statistics were calculated using an Excel template provided in Microsoft Office (Microsoft, Redmond, WA, USA), SPSS (IBM, Armonk, NY, USA) and GraphPad Prism (GraphPad Software, San Diego, CA, USA). No outliers were excluded in any of the experiments or analyses. P < 0.05 was considered statistically significant.

Twelve healthy subjects (three women, nine men) participated in the clinical study (Rysa et al., 2013). The mean age was 24 years (SD ± 5.2; range 19–38 years), the mean weight was 73 kg (SD ± 10.8; range 57–98 kg), and the mean BMI was 24.0 (SD ± 2.8; range 20.6–28.9 kg/m²).

Rifampicin 600 mg once per day for a week increased serum 4βHC concentration when compared with placebo dosing as expected (Table 1). The mean intraindividual induction was 2.2-fold (range 1.14–3.72). Also 4βHC concentration normalized by total cholesterol concentration was elevated to a similar extent. Serum 25HC and 27HC concentrations were not affected by rifampicin. Cholesterol, HDL, LDL, and triglycerides were not changed statistically significantly by rifampicin.

The qRT-PCR analyses for mRNA expression of cholesterol transporters and their regulators were performed in mononuclear cells isolated from the 12 volunteers. ABCA1 expression was induced by rifampicin dosing when analyzed with one-sample t-test (intraindividual fold induction 1.40, P = 0.042). The expression of other genes did not change significantly (Figure 1). The quantitative measurement of PXR and ABCG5 expression was unsuccessful due to low expression level, but the expression was detectable with conventional RT-PCR in all subjects. ABCG8 was not detected.

In the whole genome gene expression analysis of the 12 study subjects’ mononuclear cells with the Illumina Human HT-12 expression beads, only one gene was differentially expressed when conventional false discovery rate correction was used; ornithine decarboxylase antizyme 2 (OAZ2) was downregulated by rifampicin treatment when compared with the placebo arm. When less stringent statistical analysis was employed, the expression of 15 genes was significantly dysregulated (P < 0.05 without false discovery rate correction) by rifampicin treatment (Supplementary Table 3).

The functional cholesterol transport was studied in foam cells generated from human primary monocyte–macrophages in vitro. Experiments were performed both with and without acceptors (ApoAI and HDL₂). Macrophages were simultaneously exposed to a study compound and 50 μg/ml ³H-labeled acetylated LDL for 24 h. Cholesterol efflux was induced by 4βHC, especially when ApoAI was used as acceptor. The efflux facilitated by ApoAI was induced 1.5-fold at 20 µM 4βHC concentration (P = 0.0014,; Figure 2B). HDL₂-facilitated efflux was elevated 1.3-fold at 20 µM concentration (P = 0.078 statistically not significant; Figure 2C). Efflux to medium without acceptors was not affected. Rifampicin did not have an effect on cholesterol efflux. 22R-hydroxycholesterol used as a positive control induced cholesterol efflux as expected.

Cholesterol influx was affected by oxysterols even more pronouncedly than efflux. The 1 µM concentration of 4βHC repressed influx by 21% (P < 0.001), and 20 µM concentration caused repression of 37% (P < 0.0001) (Figure 2D). The maximal repression caused by 22RHC was 40% (P < 0.0001). While efflux to medium was significantly increased by ApoAI and HDL₂ acceptors as expected, the suppressing effect of oxysterols on the cholesterol influx was not affected by ApoAI and HDL₂ (Figures 2E, F). Rifampicin repressed cholesterol influx when normalized to protein content; this is explained by an increase in protein content elicited by rifampicin. The suppressing effect of oxysterols on influx was not affected by protein content normalization.

In in vitro experiments with human primary monocyte–macrophages (without AcLDL incubation) and foam cells (with prior AcLDL incubation), foam cells had noticeably higher expression of efflux transporters ABCA1, ABCG1, and SR-B1 when compared with macrophages (Supplementary Figure 1A). AcLDL treatment had transporter-specific effects on the influx transporters (Supplementary Figure 1B). Both LDL receptor and lectin-like oxidized LDL receptor-1 (LOX-1) were strongly repressed. The inducible degrader of the LDL receptor (IDOL), a major regulator of LDL receptor in macrophages and an LXR target (Hong et al., 2014), was induced in foam cells compared with macrophages. The expression of cluster of differentiation 36 (CD36) was induced by AcLDL treatment.

The incubation with 4βHC caused dose-dependent induction of efflux transporters ABCA1 and ABCG1 mRNAs in macrophages that reached statistical significance at 1 µM and 10 µM concentrations, respectively (Figures 3A, C). The maximum induction with 20 µM concentration of 4βHC was 2.5-fold for ABCA1 (P < 0.0001) and 4.9-fold for ABCG1 (P < 0.0001). In foam cells, ABCG1 was induced by 4βHC (maximum induction 2.0-fold, P = 0.0058), while ABCA1 was not induced (Figures 3B, D). It is noteworthy that 4βHC was able to further increase the expression of ABCG1 even in the setting of already AcLDL-induced ABCG1 expression.

The incubation with 4βHC caused dose-dependent repression of LOX-1 in macrophages (reaching statistical significance at 20 µM concentration (P = 0.016); Figure 4E). The maximum LOX-1 repression was 0.56-fold (P = 0.037) in macrophages. IDOL expression was dose-dependently induced in macrophages by 4βHC reaching statistical significance at 20 µM concentration (2.6-fold, P = 0.0012; Figure 4A). There were no effect of 4bHC on the expression of other transcripts measured (Figures 4B–D, F–J).

The incubation of macrophages with 22RHC, a positive control, induced ABCA1 and ABCG1 as expected, and also induced IDOL and repressed LDLr and LOX-1 (Figures 3 and 4). In foam cells, 22RHC induced SR-B1 (Figure 3F). We also measured the effect of rifampicin on the expression of cholesterol transporters. Rifampicin at 10 µM (but not at 20 µM) concentration caused a small decrease in SR-B1 mRNA expression in macrophages (Figure 3E). Rifampicin induced LDLr at 20 µM in macrophages and foam cells (Figure 4). LOX-1 expression was induced by rifampicin at 10 µM (but not 20 µM) concentration in macrophages and foam cells.

To further investigate the in vivo effects of PXR activation on LXR targets in peripheral tissues, rats were dosed with PCN, a rat PXR agonist, versus vehicle control for 1, 3, or 6 days. The serum concentration of 4βHC was elevated already on Day 1 (1.3-fold; 66.0 ± 13.9 vs. 85.1 ± 21.1 ng/ml, P < 0.05) and further increased on Day 6 (8.7-fold; 55.2 ± 5.8 vs. 480 ± 95 ng/ml, P < 0.05) by PCN dosing. PCN did not affect the concentration of 4α-hydroxycholesterol. The mean serum 4α-hydroxycholesterol concentration was 13.4 ± 3.0 ng/ml.

The mRNA expression of efflux and influx transporters was measured in rat peripheral tissues (Figure 5 and Supplementary Figures 2–5). ABCA1 and ABCG1 were induced by PCN in the heart (days 3 and 6, Figure 5). ABCA1 was induced in the muscle (days 1 and 6, Supplementary Figure 2). LDLr was repressed and IDOL, the negative regulator of LDLr, was induced in the heart on day 3. To interrogate the regulation of LDLr, we calculated the ratio of LDLr to IDOL (LDLr/IDOL) which was repressed by PCN dosing on day 3 in the heart (Supplementary Figure 5). In the muscle tissue, LDLr tended to be suppressed and IDOL tended to be induced while LDLr/IDOL was suppressed on days 3 and 6. Although LDLr and IDOL were not affected by PCN in white adipose tissue (Supplementary Figure 3), LDLr/IDOL ratio was repressed on day 6. In adrenal gland, LDLr was repressed on day 1 and IDOL on day 6 (Supplementary Figure 4). LDLr/IDOL ratio was downregulated on day 1 and upregulated on day 3 in adrenal gland. The expression of CD36 was induced in the heart (day 1 and day 3) and muscle (day 1), and repressed in the adrenal gland (day 6).

The protein expression of ABCA1 and ABCG1 in the left ventricle of the rat heart was measured on day 6. ABCA1 protein was induced 2.5-fold in the heart (P = 0.0009; Figure 6), while the trend of the 1.4-fold ABCG1 induction was statistically not significant (P = 0.051; Supplementary Figure 6).