High-Density Lipoprotein Reduction Differentially Modulates to Classical and Nonclassical Monocyte Subpopulations in Metabolic Syndrome Patients and in LPS-Stimulated Primary Human Monocytes In Vitro

Figure 2

Effect of HDL on LPS-stimulated primary human monocytes in vitro. Representative polyacrylamide gel showing a metabolic syndrome patient’s serum sample in which HDL was totally removed (−HDL) and then reconstituted with 0.77 mmol/L (30 mg/dL) or 1.55 mmol/L (60 mg/dL) HDL (a). As compared to untreated cells, LPS stimulation induced reduction in the CM percentage in low HDL levels (zero and 30 mg/dL) (b, left and middle panels, resp.). In contrast, the effect of LPS on the CM percentage was abolished in 60 mg/dL HDL that resembled a high HDL concentration (b, right panel). LPS stimulation did not significantly modify the IM percentage neither in low nor in high HDL concentrations (c, left, middle and right panels, resp.). As compared to untreated cells, LPS stimulation increased the NCM percentage in zero and 30 mg/dL HDL (d, left and middle panels, resp.). On the contrary, the effect of LPS on the NCM percentage was abolished in high HDL concentrations (d, right panel). As compared to untreated cells, LPS stimulation increased IL-1β production in primary human monocytes cultured in low HDL concentrations (e, left and middle panels, resp.). In contrast, the effect of LPS on IL-1β production was 1.5-fold reduced in 60 mg/dL HDL (e, right panel). Monocytes were isolated from white blood cells by CD14-positive selection using magnetic columns and placed in 0.77 mmol/L (30 mg/dL) or 1.55 mmol/L (60 mg/dL) HDL-enriched culture media (1 × 10⁶ monocytes per well), in the presence or absence of gram-negative bacteria-derived LPS at 1 μg/mL for six hours at 37°C. After this time, monocytes were incubated with anti-CD14 PE/Cy7 and anti-CD16 FITC as described. For the gating strategy, untreated and LPS-treated cells were firstly gated for singlets on a FSC-H/FSC-A density plot. On the monocyte gate, living untreated and LPS-treated cells were further gated using the Live/Dead Aqua stain. Living monocytes were then gated to determine CD14- and CD16-positive expression and identify monocyte subpopulations as follows: CD14^highCD16⁻, classical monocytes; CD14^highCD16⁺, intermediate monocytes; and CD14^lowCD16⁺, nonclassical monocytes. In (b–e), data are expressed as mean ± standard deviation. Significant differences were considered when .