Low-density lipoprotein (LDL)-dependent uptake of Gram-positive lipoteichoic acid and Gram-negative lipopolysaccharide occurs through LDL receptor

Abstract

Lipoteichoic acid (LTA) and lipopolysaccharide (LPS) are bacterial lipids that stimulate pro-inflammatory cytokine production, thereby exacerbating sepsis pathophysiology. Proprotein convertase subtilisin/kexin type 9 (PCSK9) negatively regulates uptake of cholesterol by downregulating hepatic lipoprotein receptors, including low-density lipoprotein (LDL) receptor (LDLR) and possibly LDLR-related protein-1 (LRP1). PCSK9 also negatively regulates Gram-negative LPS uptake by hepatocytes, however this mechanism is not completely characterized and mechanisms of Gram-positive LTA uptake are unknown. Therefore, our objective was to elucidate the mechanisms through which PCSK9 regulates uptake of LTA and LPS by investigating the roles of lipoproteins and lipoprotein receptors. Here we show that plasma PCSK9 concentrations increase transiently over time in septic and non-septic critically ill patients, with highly similar profiles over 14 days. Using flow cytometry, we demonstrate that PCSK9 negatively regulates LDLR-mediated uptake of LTA and LPS by HepG2 hepatocytes through an LDL-dependent mechanism, whereas LRP1 and high-density lipoprotein do not contribute to this uptake pathway. Bacterial lipid uptake by hepatocytes was not associated with cytokine production or hepatocellular injury. In conclusion, our study characterizes an LDL-dependent and LDLR-mediated bacterial lipid uptake pathway regulated by PCSK9, and provides evidence in support of PCSK9 inhibition as a potential therapeutic strategy for sepsis.

Trial registration: ClinicalTrials.gov NCT01355042.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1

Plasma PCSK9 concentrations in septic and non-septic ICU patients over time compared to healthy controls. Plasma for healthy controls was collected on a single day (mean PCSK9 concentrations of 146.4 ± 13.4 ng/mL). Data are expressed as mean ± SEM, *p 

Figure 2

Time-course and dose-response of LTA uptake by HepG2 cells over 24 h. Uptake of fluorescent LTA by cells cultured in 20% normal human serum was visualized through fluorescence microscopy in untreated cells (A) or in cells treated with 10 μg/mL of BODIPY 630/650-LTA for 2 h (B), 6 h (C), or 24 h (D), or measured via flow cytometry for cells cultured in either 20% normal serum or lipoprotein-deficient serum (prepared in laboratory) and treated with increasing concentrations of fluorescent LTA over 24 h (E). Nuclei are stained in blue with DAPI, and BODIPY 630/650-LTA appears red (200× magnification). Data are representative of 3 experiments (A–D), and are expressed as mean ± SEM from 3 experiments (E). ***p < 0.001 by two-way ANOVA. MFI, mean fluorescence intensity.

Figure 3

PCSK9 regulates lipoprotein-dependent uptake of LTA and LPS by HepG2 cells through LDLR, not LRP1. HepG2 cells were cultured in 20% normal human serum (A,C) or (commercially available) lipoprotein-deficient human serum (B,D) and were pre-treated with 2.5 μg/mL of recombinant human PCSK9 or vehicle control at 6 h before, as well as anti-LDLR, anti-LRP1, or control IgG antibodies at 2 h before treatment with BODIPY 630/650-LTA (10 μg/mL; A,B) or AlexaFluor 488-LPS (2.5 μg/mL; C,D) for 24 h. Data were collected from 4–5 experiments at 24 hours after LPS or LTA treatment using flow cytometry, and are expressed as geometric mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA. MFI, mean fluorescence intensity.

Figure 4

Dose-response of LDL or HDL add-back to normal or lipoprotein-deficient serum on the uptake of LTA and LPS by HepG2 cells. Cells were cultured in 20% lipoprotein-deficient serum (prepared in laboratory) and treated with 2.5 μg/mL of BODIPY 630/650-LTA (A) or 2.5 μg/mL of AlexaFluor 488-LPS (B) over 24 h in the presence of increasing concentrations of LDL or HDL. Data are shown as mean ± SEM from 3–4 experiments. **p < 0.01, ***p < 0.001 vs. 0 μg/mL of LDL by one-way ANOVA. LPDS, lipoprotein-deficient serum; MFI, mean fluorescence intensity.

Figure 5

LDL-dependent uptake of LTA and LPS by HepG2 cells is mediated by LDLR. Cells were cultured in 20% normal human serum or donor-matched lipoprotein-deficient serum (prepared in laboratory), with or without 100 μg/mL of LDL or HDL, and treated with anti-LDLR antibody or control IgG for two hours prior to 24 h treatment with 2.5 μg/mL of BODIPY 630/650-LTA (A) or AlexaFluor 488-LPS (B). Data are presented as geometric mean fluorescence intensity ± SEM from 3–4 experiments, and were normalized by subtracting background auto-fluorescence of untreated cells. Different letters indicate significant differences between groups with p < 0.01 by ANOVA. MFI, mean fluorescence intensity.