CETP Lowers TLR4 Expression Which Attenuates the Inflammatory Response Induced by LPS and Polymicrobial Sepsis

Abstract

Sepsis is a systemic inflammatory response to infection eliciting high mortality rate which is a serious health problem. Despite numerous studies seeking for therapeutic alternatives, the mechanisms involved in this disease remain elusive. In this study we evaluated the influence of cholesteryl ester transfer protein (CETP), a glycoprotein that promotes the transfer of lipids between lipoproteins, on the inflammatory response in mice. Human CETP transgenic mice were compared to control mice (wild type, WT) after polymicrobial sepsis induced by cecal ligation and puncture (CLP), aiming at investigating their survival rate and inflammatory profiles. Macrophages from the peritoneal cavity were stimulated with LPS in the presence or absence of recombinant CETP for phenotypic and functional studies. In comparison to WT mice, CETP mice showed higher survival rate, lower IL-6 plasma concentration, and decreased liver toll-like receptor 4 (TLR4) and acyloxyacyl hydrolase (AOAH) protein. Moreover, macrophages from WT mice to which recombinant human CETP was added decreased LPS uptake, TLR4 expression, NF-κB activation and IL-6 secretion. This raises the possibility for new therapeutic tools in sepsis while suggesting that lowering CETP by pharmacological inhibitors should be inconvenient in the context of sepsis and infectious diseases.

Figure 1

Human CETP expressing mice are more resistant to polymicrobial sepsis than wild type mice. CETP and WT mice submitted to CLP and control mice were sham-operated on (n = 8). Survival rate study of mice monitored every 8 hours for 5 days after CLP. ^∗ p = 0.0267 by a log-rank test between CLP groups. Mortality rate in the sham group did not change.

Figure 2

Plasma IL-6 levels in CETP and WT mice after CLP induced polymicrobial sepsis. Plasma IL-6 levels were measured at 24 h and 48 h after CLP by ELISA. Results are expressed as means ± SD, n = 6 mice per group. Data were analyzed by one-way ANOVA (p = 0.008: WT 48 h versus CETP 48 h; p = 0.004: CETP 24 h versus CETP 48 h).

Figure 3

The hepatic content of TLR4 and AOAH decreased in the liver from CETP as compared to WT mice. Liver samples were harvested at 24 h and 48 h after CLP. TLR4 ((a) and (b)) as well as AOAH ((c) and (d)) protein expression was determined by Western blotting. Graph presented as mean ± SD of arbitrary units corrected by β-actin level ((a) and (b)); n = 3–5, differences by one-way ANOVA, and p < 0.05. ((b) and (d)) Representative Western blot images of TLR4, AOAH, and β-actin protein expression.

Figure 4

Confocal microscopy of macrophages: colocalization between LPS and CETP (magnification of 600x). Representative confocal image of peritoneal macrophages from WT mice stimulated with LPS (1 µg/mL) for 4 h in the presence of the LBP, known to be attached to LPS: LBP was utilized as a positive control (a) or CETP (b) (1 µg/mL). FITC-LPS, green; nucleus, blue (DAPI); LBP, yellow; CD68⁺ macrophages, red; CETP, blue (in the absence of DAPI because CETP antibody is also blue); merge: colocalization between markers.

Figure 5

CETP reduces macrophage LPS uptake after LPS stimulus. Peritoneal macrophages obtained from WT mice were stimulated for 24 h with LPS-Alexa Fluor® 488 (1 µg/mL) in the absence or presence of CETP (0.1, 0.5, and 1.0 µg/mL). (a) Representative FACS dot plots gated on F4/80⁺ cells (macrophages only) and the percentage of LPS-Alexa 488⁺ cells are shown. (b) Representative flow cytometry histograms comparing macrophages (F4/80⁺) from WT and respective CETP groups stimulated with LPS. (c) Graph presented as mean ± SD of MFI (median fluorescence intensity) analyzed using FACSDiva software (n = 4) representative of three independent experiments, by one-way ANOVA p = 0.0014 and posttest (Newman-Keuls Multiple Comparison Test; ^∗ p < 0.05 and ^# p < 0.01).

Figure 6

CETP reduces TLR4 expression in LPS-stimulated macrophages. Peritoneal macrophages obtained from WT mice were stimulated for 24 h with LPS-Alexa Fluor 488 (1 µg/mL) in the absence or presence of CETP (0.1, 0.5, and 1.0 µg/mL). (a) Representative FACS dot plots gated on F4/80⁺ cells (macrophages only) and the percentage of TLR4⁺ cells are shown. (b) Representative flow cytometry histograms of TLR4 comparing macrophages (F4/80⁺) from WT and respective CETP groups stimulated with LPS. (c) Graph presented as mean ± SD of MFI (median fluorescence intensity) analyzed using FACSDiva software (n = 4), representative of three independent experiments, by one-way ANOVA p = 0.0014 and posttest (Newman-Keuls Multiple Comparison Test; ^∗ p < 0.05 and ^# p < 0.01).

Figure 7

CETP reduces LPS-stimulated transcription factor NF-κB activation. Peritoneal macrophages obtained from WT mice were stimulated for 24 h with LPS-Alexa Fluor 488 (1 µg/mL) in the absence or presence of CETP (0.1, 0.5, and 1.0 µg/mL). (a) Representative FACS dot plots gated on F4/80⁺ cells (macrophages only) and the percentage of NF-κB ⁺cells are shown. (b) Representative flow cytometry histograms of NF-κB comparing macrophages (F4/80⁺) from WT and respective CETP groups stimulated with LPS. (c) Graph presented as mean ± SD of MFI (median fluorescence intensity) analyzed using FACSDiva software (n = 4) representative of three independent experiments, by one-way ANOVA p = 0.0019 and posttest (Newman-Keuls Multiple Comparison Test; ^∗ p < 0.05 and ^# p < 0.01).

Figure 8

CETP reduces the release of IL-6 levels in LPS-stimulated macrophages. Peritoneal macrophages from WT mice were stimulated for 24 h with LPS-Alexa Fluor 488 (1 µg/mL) in the absence or in the presence of different concentrations of recombinant human CETP (0.1, 0.5, and 1.0 µg/mL). IL-6 concentration release to cell culture medium decreased dose dependently in the presence of CETP (n = 4). Graph presented as mean ± SD representative of three independent experiments. Data were analyzed by one-way ANOVA p = 0.0014 and posttest (Newman-Keuls Multiple Comparison Test; ^∗ p < 0.05: control LPS versus CETP group; and ^# p < 0.01: CETP 0.1 versus CETP 1.0).

Figure 9

The presence of CETP reduced the cellular uptake of LPS, TLR4 expression, and NF-κB activation (p65) and consequently decreased the secretion of IL-6 indicating that CETP interferes in TLR4 signaling pathways.