Fig 1.
Time course of LPS uptake by hepatocytes.
(A) For a live-imaging experiment fresh isolated primary hepatocytes were plated on 35 mm glass bottom dish precoated with collagen I (MatTek) and incubated overnight. The cells were stained with Hoechst to identify nuclei (blue) prior Alexa Fluor 488-conjugated E.coli LPS (green) addition and then immediately imaged constantly with the 63x objective using a Leica Inverted Fluorescence microscope, at 37°C and 5% CO2. (B) Time course of LPS uptake by HepG2 cells. HepG2 cells were treated with Alexa-488 FluorTM E.coli LPS (2.5 μg/ml) for 15, 30 min, 1, 2, 4 and 6 hours in media containing 20% human plasma. LPS uptake was measured by flow cytometry. Data presented as mean fluorescence intensity, mean±SEM (n = 3).
Fig 2.
Ldlr knockout (Ldlr-/-) reduces plasma clearance of LPS and hepatic LPS uptake.
(A) LPS levels in plasma were measured by microplate reader 5 min, 1 and 6 hours post injection of a non-lethal dose of FITC-conjugated E.coli LPS (5 mg/kg) into tail vein of control (wild type) and Ldlr-/- mice. (B) Data are presented as mean fluorescence intensity of homogenized liver tissue detected by the microplate reader and normalized to mice injected with non-fluorescent LPS and tissue weight, mean±SEM, (n = 5). Analyzed by two-way ANOVA.
Fig 3.
LPS uptake into hepatocytes is LDLR-dependent.
(A) Primary hepatocytes were isolated from wild type and Ldlr-/- mice, plated on 12 mm coverslips precoated with Collagen I in DMEM medium with 20% Bovine Serum, 100 units/mL penicillin, and 100 units/mL streptomycin. Cells were allowed to attach to coverslips for 6–12 hours prior the treatment with 2.5 ug/mL Alexa Fluor 488–conjugated LPS was added for 1, 6 and 24 hours total. Coverslips were fixed with 4% paraformaldehyde for 20 min and stained with DAPI to identify nuclei (blue). Images are representative of three independent experiments. (B) 2D image of primary hepatocytes isolated form WT mice and treated with 2.5 μg/mL Alexa Fluor 488–conjugated LPS for 24 hours. LPS uptake was visualized using Leica Inverted Fluorescence microscope (x63 magnification) and analyzed using VOLOCITY software (PerkinElmer Inc.).
Fig 4.
HepG2 cells clear LPS in LDLR-dependent manner.
(A) The LDLR specific and scrambled siRNAs were reversely transfected into HepG2 cells. After 24, 48 and 72 hrs, the cells were detached and stained with LDLR-PE conjugated antibody. The LDLR levels were analyzed by flow cytometry. Data presented as percent of mean fluorescence intensity from scrambled control, mean±SEM (n = 3). Highest knockdown efficiency (73%) was achieved 48 hours after transfection. (B) Effect of LDL receptor knockdown on LPS uptake. HepG2 cells were reversely transfected with siRNA targeting LDLR and control scrambled siRNA. Cells were treated with Alexa Fluor 488-conjugated LPS (2.5 μg/ml) 48 hours after transfection for 24 hours. LPS uptake was measured by flow cytometry. Data presented as mean fluorescence intensity, mean±SEM (n = 3). (C) PCSK9 incubation decreased LPS uptake by HepG2 cells. Scrambled siRNA was reversely transfected into HepG2 cells, than treated similarly as described above with PCSK9 as a complementary approach to decreasing LDLR. Data presented as mean fluorescence intensity, mean±SEM (n = 3). (D) HepG2 cells uptake LDL-labeled LPS complex via LDLR. Freshly isolated LDL form healthy donors was labeled with Alexa-488 Fluor LPS, and separated with the Sephadex column. HepG2 cells were pretreated with PCSK9 (3 μg/mL) or LDLR AB (3 μg/mL) to block the LDLR levels before adding LDL-LPS complex (50 μg/mL). The LDL-LPS uptake was analyzed by flow cytometry. Data presented as mean fluorescence intensity, mean±SEM (n = 3).
Fig 5.
LPS uptake is mediated by the LDLR in human hepatocytes.
(A) PCSK9 further reduced LPS uptake in Ldlr-/- cells. Primary hepatocytes were isolated from Ldlr-/- mice. Cells were allowed to attach to coverslips for 6–12 hours than treated with 2.5 ug/mL Alexa Fluor 488–conjugated LPS for 6 hours total. Coverslips were fixed with 4% paraformaldehyde for 20 min and stained with DAPI to identify nuclei (blue). LPS uptake was visualized using Leica Inverted Fluorescence microscope (x63 magnification) and analyzed with VELOCITY software. Images are representative of three independent experiments. (B) Ldlr-/- mice have increased expression of VLDLR in a liver. VLDR receptor protein expression by Western blot in wild type and Ldlr-/- mouse liver at time points 1 hour and 6 hours after injection of LPS. Data presented as mean±SEM, (***P = 0.0007, compared with control 1h; **P = 0.0026, compared with control 6h, n = 3). (C) PCSK9 further altered uptake of LPS in HepG2 cells with LDLR knockdown. HepG2 cells were reversely transfected with siRNA targeting LDLR and control scrambled siRNA. Recombinant human PCSK9 (3 μg/ml) was added 4 hours before and 4 and 19 hours after LPS treatment. Cells were treated with Alexa Fluor 488-conjugated LPS (2.5 μg/ml) 48 hours after transfection for 24 hours. LPS uptake was measured by flow cytometry. Data presented as mean fluorescence intensity, mean±SEM (n = 3).