Systematic RNA-interference in primary human monocyte-derived macrophages: A high-throughput platform to study foam cell formation

Abstract

Macrophage-derived foam cells are key regulators of atherogenesis. They accumulate in atherosclerotic plaques and support inflammatory processes by producing cytokines and chemokines. Identifying factors that regulate macrophage lipid uptake may reveal therapeutic targets for coronary artery disease (CAD). Here, we establish a high-throughput screening workflow to systematically identify genes that impact the uptake of DiI-labeled low-density lipoprotein (LDL) into monocyte-derived primary human macrophages. For this, monocytes isolated from peripheral blood were seeded onto 384-well plates, solid-phase transfected with siRNAs, differentiated in vitro into macrophages, and LDL-uptake per cell was measured by automated microscopy and quantitative image analysis. We applied this workflow to study how silencing of 89 genes impacts LDL-uptake into cells from 16 patients with CAD and 16 age-matched controls. Silencing of four novel genes (APOC1, CMTM6, FABP4, WBP5) reduced macrophage LDL-uptake. Additionally, knockdown of the chemokine receptor CXCR4 reduced LDL-uptake, most likely through a G-protein coupled mechanism that involves the CXCR4 ligand macrophage-induced factor (MIF), but is independent of CXCL12. We introduce a high-throughput strategy to systematically study gene function directly in primary CAD-patient cells. Our results propose a function for the MIF/CXCR4 signaling pathway, as well as several novel candidate genes impacting lipid uptake into human macrophages.

Figure 1

Microscope-based screening platform for solid-phase siRNA-transfection and functional analyses in primary human monocyte-derived macrophages. (a) Workflow illustrating the experimental setup: Blood sampling, monocyte isolation and seeding on siRNA-coated plates, monocyte-macrophage differentiation and solid-phase siRNA transfection, functional assays, image acquisition and analysis. (b,c) Representative images reflecting siRNA transfection efficiency as monitored with Cy3-labelled non-silencing control-siRNA (b, red) and macrophage marker CD68 (c, green) in monocyte-derived macrophages three days after siRNA transfection. (d) Representative images of DiI-LDL signals in cells transfected with either control siRNA or siRNA against CSF1R. (e,f) Relative DiI signal intensities in macrophages from 6 healthy individuals treated with control or CSF1R-siRNA as measured through quantitative image analysis. Each dot in (e) indicates the mean cellular DiI signal intensity per 1 frame from 15 technical replicates per individual (i.e., independent wells of a plate). (f) Shows change in DiI signal relative to negative control in percent (±s.d.). (g) Mean±s.d. relative change in DiI signal compared to negative control in percent for siRNAs against LDLR and CD36. Macrophages from 6 healthy individuals were used with 12 technical replicates per individual. P values were calculated with Bonferroni corrected unpaired two-tailed t-test. **P < 0.001; ***P < 0.0001.

Figure 2

Knockdown of APOC1, CMTM6, FABP4 and WBP5 reduces DiI-LDL uptake into primary human monocyte-derived macrophages. Bars reflect mean ± S.D relative change in cellular DiI-LDL fluorescence signal intensities as determined from cultured primary human monocyte-derived macrophages during the replication screen. Cells were solid-phase transfected with siRNAs against indicated genes (2 independent siRNAs/gene), and DiI-LDL uptake was measured relative to control siRNA treated cells with 1.0 being the mean intensity of all negative control wells for each plate. Shown are results from cells of 10 healthy individuals (grey) and 10 patients with coronary artery disease (orange). To determine significance, deviation values (Dev; see Methods) were calculated for each siRNA tested in each individual relative to the mean intensity of respective negative control wells, and the median deviation was calculated for the control group (Md(C)), the patient group (Md(P)), and the combination of both groups (Md(C + P)) separately. Finally, the mean median deviation shown was calculated from the median deviation of results from three technical replicates per individual (i.e, three independent parallel analyses of cells isolated from the same blood draw). **Dev <−1.0 for at least 2 siRNAs in 1 group (controls, patients, controls + patients). For full siRNA screening results from primary and replication screens, see Supplemental Data.

Figure 3

The MIF-CXCR4 signaling axis regulates DiI-LDL uptake into primary human monocyte-derived macrophages. (a) Relative change in DiI signal intensity compared to negative control (in percent) in macrophages treated with siRNAs against CXCR4 or CXCR7 (n = 5). (b) Representative images of DiI-LDL signal in macrophages treated with CXCR4- or control-siRNA. (c) Relative change in DiI signal intensity compared to negative control in per cent in macrophages treated with siRNAs against CXCL12 or MIF (n = 3). (d) Relative change in DiI fluorescence intensity compared to negative control (untreated cells) after stimulation with either AMD3100 (AMD) alone for 8 or 24 hours, or CXCL12 (1 μg/ml) or MIF (1 μg/ml) for 8 or 24 hours in the absence or presence of AMD (n = 3). (e) Relative change in DiI fluorescence intensity compared to negative control siRNA after treatment with CXCR4-siRNA in the absence or the presence of MIF (1 μg/ml) for 24 hours. (f) Relative change in DiI fluorescence intensity compared to negative control (untreated cells) after stimulation with MIF (1 μg/ml) in the absence or presence of pertussis toxin (250 ng/ml) for 24 hours (n = 3). P-values were calculated with unpaired, two-tailed t-test followed by a post-hoc Dunn’s multiple comparison test. ***P < 0.0001.