Fig 1.
LPS uptake is mediated by endocytosis in RAW264.7 cells.
Cells were treated with fluorescence-labelled LPS and analysed by flow cytometry. (A) Representative histogram of fluorescence intensity for Alexa Fluor 594-labelled LPS in RAW264.7 cells treated with medium alone (Untreated; greyscale) and Alexa Fluor 594-labelled LPS at 1 μg/mL for 4 h (red line). (B) Changes in mean fluorescence intensity (MFI) of Alexa Fluor 594 in RAW264.7 cells treated with Alexa Fluor 594-labelled LPS at concentrations ranging from 1 ng/mL to 1 μg/mL for 4 h (n = 3 means the number of independent experiments, Dunnett’s test). (C) Time course of MFI in RAW264.7 cells treated with Alexa Fluor 594-labelled LPS at 1 μg/mL (n = 3, Dunnett’s test). (D) Representative images of LPS uptake visualised by Alexa Fluor 488-labelled LPS (green) in RAW264.7 cells. Scale bars: 50 μm. (E) MFI of Alexa Fluor 594-labelled LPS at 1 μg/mL in RAW264.7 cells treated with endocytosis inhibitors for 1 h (n = 3, Tukey’s test). Each column represents the MFIs of Alexa Fluor 594-labelled LPS relative to the concentration at “0” or medium alone (Unt), which was arbitrarily defined as 100%. Data are presented as the means ± SEM. MFI; mean fluorescence intensity. Unt; untreated.
Fig 2.
Involvement of CD14 on LPS uptake and CXCL10 release in response to LPS.
(A) Time course of MFI related to CD14 in RAW264.7 cells treated with LPS at 1 μg/mL (n = 3, Dunnett’s test). (B) Effect of anti-CD14 antibody at 20 μg/mL on LPS uptake (n = 3, Tukey’s test). Neutralising antibody was added 1 h before treatment with Alexa Fluor 594-labelled LPS at 1 μg/mL and incubated for 4 h. MFI relative to the untreated group (medium alone) was arbitrarily defined as 100%. (C) Time course of CXCL10 levels in the culture supernatants obtained from cells treated with LPS at 1 μg/mL (n = 6, Dunnett’s test). (D) Effect of anti-CD14 antibody at 20 μg/mL on CXCL10 release from RAW264.7 cells treated with LPS at 1 μg/mL (n = 3, Tukey’s test). CXCL10 levels in the culture supernatants obtained from cells treated with LPS and/or anti-CD14 antibody for 24 h were measured by ELISA. Data are presented as the means ± SEM. MFI; mean fluorescence intensity. Unt; untreated. Ab; antibody.
Fig 3.
Inhibitory effect of AGE-3 on the cellular response to LPS in RAW264.7 cells.
(A) Representative histogram of fluorescence intensity for Alexa Fluor 594-labelled LPS in RAW264.7 cells treated with medium alone (Untreated; greyscale), Alexa Fluor 594-labelled LPS at 1 μg/mL (red line), and a combination of Alexa Fluor 594-labelled LPS and AGE-3 at 200 μg/mL (blue line). (B) Effect of AGE-3 on LPS uptake (n = 3, Tukey’s test). Cells were concomitantly treated with Alexa Fluor 594-labelled LPS at 1 μg/mL and AGE-3 at 200 μg/mL for 4 h. MFI relative to the untreated group (medium alone) was arbitrarily defined as 100%. (C) Representative images of LPS uptake visualised by Alexa Fluor 488-labelled LPS (green) in RAW264.7 cells treated with AGE-3 at 200 μg/mL for 4 h. Scale bars: 50 μm. (D) Time course of MFI related to CD14 in RAW264.7 cells treated with AGE-3 at 200 μg/mL (n = 3, Dunnett’s test). (E) Changes in MFI related to CD14 in RAW264.7 cells treated with AGE-3 at concentrations ranging from 2 to 200 μg/mL for 4 h (n = 3, Dunnett’s test). (F) Effect of AGE-3 on the time course of changes in CD14 expression in RAW264.7 cells treated with LPS at 1 μg/mL (n = 5, Dunnett’s test). (G) Effect of AGE-3 at 200 μg/mL on CXCL10 release from RAW264.7 cells treated with LPS at 1 μg/mL (n = 5, Tukey’s test). CXCL10 levels in the culture supernatants obtained from cells treated with LPS and/or AGE-3 for 24 h were measured by ELISA. Data are presented as the means ± SEM. Unt; untreated.
Fig 4.
Involvement of RAGE on the inhibitory effect of AGE-3.
(A) Effect of pharmacological inhibitors on the AGE-3-induced downregulation of CD14 expression (n = 3; AGE-3+LPS-RS and AGE-3+Anti-SR-A Ab, n = 6; Unt, AGE-3 and AGE-3+FPS-ZM1 at 0.25–1.0 μM, Tukey’s test). Cells were concomitantly treated with FPZ-ZM1 at 1 μM or LPS-RS at 10 μg/mL and AGE-3 at 200 μg/mL for 4 h. (B) Effect of anti-RAGE neutralising antibody on the AGE-3-induced downregulation of CD14 expression (n = 3). Cells were pre-treated with anti-RAGE antibody at 20 μg/mL for 1 h before incubation with AGE-3 for 4 h. (C) Effect of FPS-ZM1 on LPS uptake in RAW264.7 cells treated with AGE-3 (n = 9, Tukey’s test). Cells were concomitantly treated with Alexa Fluor 594-labelled LPS at 1 μg/mL, AGE-3 at 200 μg/mL, and/or FPS-ZM1 at 1 μM for 4 h. (D) Effect of anti-RAGE neutralising antibody on LPS uptake in RAW264.7 cells. Cells were pre-treated with anti-RAGE antibody at 20 μg/mL for 1 h before incubation with Alexa Fluor 594-labelled LPS and AGE-3 for 4 h. MFI relative to the untreated group (medium alone) was arbitrarily defined as 100%. (E) Effect of FPS-ZM1 at 1 μM and anti-RAGE neutralising antibody on CXCL10 release from RAW264.7 cells treated with LPS at 1 μg/mL and/or AGE-3 at 200 μg/mL (n = 8, Tukey’s test). CXCL10 levels in the culture supernatants obtained from cells treated with LPS, AGE-3, FPS-ZM1 and/or anti-RAGE neutralising antibody for 24 h were measured by ELISA. (F) Effect of anti-RAGE neutralising antibody on CXCL10 release from RAW264.7 cells treated with LPS at 1 μg/mL and/or AGE-3 at 200 μg/mL (n = 5, Tukey’s test). CXCL10 levels in the culture supernatants obtained from cells treated with LPS, AGE-3 and/or anti-RAGE neutralising antibody for 24 h were measured by ELISA. Data are presented as the means ± SEM.Unt; untreated. Ab; antibody.
Fig 5.
Model depicting the inhibitory effect of AGE-3 on the response to LPS in macrophages.
LPS binding to CD14 is endocytosed via the formation of a dimerised TLR4/MD-2 complex. Subsequently, this assembly activates TRIF signalling on endosomes and the transcription of IFN-inducible genes including CXCL10. AGE-3 induces the downregulation of CD14 expression though RAGE. Downregulation of CD14 expression induced by AGE-3 contributes to the decreased LPS uptake and CXCL10 release in macrophages.