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, 118 (1), 364-75

SR-BI Protects Against Endotoxemia in Mice Through Its Roles in Glucocorticoid Production and Hepatic Clearance

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SR-BI Protects Against Endotoxemia in Mice Through Its Roles in Glucocorticoid Production and Hepatic Clearance

Lei Cai et al. J Clin Invest.

Abstract

Septic shock results from an uncontrolled inflammatory response, mediated primarily by LPS. Cholesterol transport plays an important role in the host response to LPS, as LPS is neutralized by lipoproteins and adrenal cholesterol uptake is required for antiinflammatory glucocorticoid synthesis. In this study, we show that scavenger receptor B-I (SR-BI), an HDL receptor that mediates HDL cholesterol ester uptake into cells, is required for the normal antiinflammatory response to LPS-induced endotoxic shock. Despite elevated plasma HDL levels, SR-BI-null mice displayed an uncontrollable inflammatory cytokine response and a markedly higher lethality rate than control mice in response to LPS. In addition, SR-BI-null mice showed a lack of inducible glucocorticoid synthesis in response to LPS, bacterial infection, stress, or ACTH. Glucocorticoid insufficiency in SR-BI-null mice was due to primary adrenal malfunction resulting from deficient cholesterol delivery from HDL. Furthermore, corticosterone supplementation decreased the sensitivity of SR-BI-null mice to LPS. Plasma from control and SR-BI-null mice exhibited a similar ability to neutralize LPS, whereas SR-BI-null mice showed decreased plasma clearance of LPS into the liver and hepatocytes compared with normal mice. We conclude that SR-BI in mice is required for the antiinflammatory response to LPS-induced endotoxic shock, likely through its essential role in facilitating glucocorticoid production and LPS hepatic clearance.

Figures

Figure 1
Figure 1. Hyperinflammatory response in SR-BI–/– mice challenged with LPS.
SR-BI–/– and SR-BI+/+ mice were injected i.p. with 0.5 μg/g body weight of LPS. Plasma TNF-α and IL-6 levels were determined by ELISA (A and B). Liver TNF-α and IL-6 mRNA levels were determined by Q-PCR (C and D). Values shown are the mean ± SD (n = 3). Similar results were found in 4 independent experiments.
Figure 2
Figure 2. SR-BI–/– mice are more sensitive to LPS-induced lethality.
(A) Survival of SR-BI–/– mice in response to LPS. SR-BI–/–, SR-BI+/–, and SR-BI+/+ mice (n = 8) were injected i.p. with 5 μg/g body weight of LPS. Mice were carefully monitored, and survival rates at the indicated times were recorded (SR-BI–/– vs. SR-BI+/+; P < 0.001). (B) Plasma cytokine levels in SR-BI–/– and SR-BI+/+ mice challenged with LPS. LPS (5 μg/g body weight) was injected i.p. into SR-BI+/+ and SR-BI–/– mice, and blood was collected 2 hours following LPS injection. Plasma IL-6 and TNF-α levels were determined by ELISA. Values shown are the mean ± SD (n = 4) (*P < 0.05; **P < 0.01). Similar results were seen in 3 independent experiments.
Figure 3
Figure 3. Plasma corticosterone levels are not induced in SR-BI–/– mice challenged with LPS.
(A) Corticosterone levels in SR-BI–/– and CD36–/– mice injected with various amounts of LPS (0.17, 0.5, and 1.5 μg/g body weight) and sacrificed 2 hours later. Values shown are the mean ± SD (n = 4). a, b, c, P < 0.05. (B) SR-BI–/– and SR-BI+/+ mice were given corticosterone (100 μg/ml in 0.1% ethanol) in drinking water (SR-BI–/–/C, SR-BI+/+/C) or 0.1% ethanol only 8 hours prior to i.p. injection of LPS (5 μg/g body weight). Survival rates at the indicated times following LPS administration are shown (n = 7). (C) Plasma corticosterone levels in the surviving mice. Surviving mice were sacrificed 66 hours after LPS challenge. Values shown are the mean ± SD (n = 4 for SR-BI–/–; n = 7 for SR-BI+/+). (D) Plasma TNF-α levels 2 hours after LPS injection with or without corticosterone supplementation. SR-BI+/+ and SR-BI–/– mice were injected i.p. with 0.5 and 5 μg/g body weight of LPS. Values shown are the mean ± SD (n = 4). *P < 0.05; **P < 0.01. Similar results were found in 3 independent experiments.
Figure 4
Figure 4. Primary adrenal deficiency in SR-BI–/– mice.
(A) Plasma ACTH levels in SR-BI+/+ and SR-BI–/– male mice challenged with LPS (0.5 μg/g body weight of LPS or saline) for 2 hours (B) ACTH-induced corticosterone response in SR-BI+/+ and SR-BI–/– mice. Male SR-BI+/+ and SR-BI–/– mice were injected with 2 U of ACTH s.c. and sacrificed under anesthesia at the indicated times. (C) Adrenal insufficiency in SR-BI–/– mice under stress. Female SR-BI+/+ and SR-BI–/– mice were stressed by a 3-minute cold water (5°C) swim followed by a 17-minute rest at room temperature. Values shown are mean ± SD (n = 4). Similar results were found in 3 independent experiments. *P < 0.05; **P < 0.01.
Figure 5
Figure 5. Hyperinflammatory response in SR-BI–/– mice challenged with E. coli.
SR-BI–/– and SR-BI+/+ mice were injected i.p. with 2 × 107 CFU E. coli per mouse. Two hours after the injection, mice were sacrificed and plasma was collected. (A) Plasma cytokine levels determined by ELISA. (B) Plasma corticosterone levels determined by RIA. Values shown are mean ± SD (n = 4). Similar results were found in 2 independent experiments. *P < 0.05; **P < 0.01.
Figure 6
Figure 6. Effects of adrenalectomy on LPS-induced response in SR-BI–/– and SR-BI+/+ mice.
Mice were adrenalectomized and allowed to recover for 1 week. Adrenalectomized mice (ADX) and sham-operated mice (sham) were injected i.p. with LPS (0.125 μg/g body weight), and 2 hours later, plasma cytokines and corticosterone levels were determined. (A) Plasma TNF-α levels. (B) Plasma IL-6 levels. (C) Plasma MCP-1 levels. (D) Plasma corticosterone levels. Values shown are mean ± SD (n = 5). *P < 0.05; **P < 0.01. Similar results were found in 3 independent experiments.
Figure 7
Figure 7. Adrenal gene expression during LPS-induced inflammatory response.
SR-BI+/+ and SR-BI–/– mice were injected i.p. with 0.5 μg/g body weight of LPS. Mice were sacrificed at indicated time points, and adrenals were collected for mRNA extraction and Q-PCR. (A) Adrenal StAR mRNA. (B) Adrenal Cyp11A1 mRNA. (C) Adrenal StAR Western blot (10 μg cell protein/lane). Mice were sacrificed 2 hours after LPS injection. Adrenals were collected, and proteins were extracted. (D) Adrenal LDLR mRNA. (E) Adrenal HMG-CoA reductase mRNA. Values shown are mean ± SD (n = 3) of the ratio of genes for the target genes to that of the 18S rRNA. Similar results were found in 2 independent experiments.
Figure 8
Figure 8. SR-BI–dependent uptake of LPS in hepatocytes.
(A) Primary hepatocytes were washed and incubated with 5 μg/ml of Alexa Fluor–LPS at 37°C for 1 hour in DMEM medium containing 0.5% BSA. Fluorescence microscopy was performed at ×40 original magnification with equal exposures (Alexa Fluor–LPS, red; DAPI, blue, nuclear stain). (B) Association of lipid-free and HDL-bound 125I-LPS with SR-BI+/+ and SR-BI–/– primary hepatocytes. Where indicated (LPS + HDL), 125I-LPS (5 μg) was preincubated with human HDL3 (10 μg) at room temperature for 30 minutes before addition to cells. Hepatocytes were incubated with 5 μg/ml 125I-LPS or 125I-LPS + HDL at 37°C for 1 hour in DMEM medium containing 0.5% BSA. Values shown are the mean ± SD of triplicate determinations (*P < 0.05). Similar results were found in 4 independent experiments.
Figure 9
Figure 9. LPS distribution in the liver.
(A) LPS distribution in hepatocytes and nonhepatocytes. 125I-LPS (0.125 μg/g body weight) was injected through the tail vein into SR-BI–/– and SR-BI+/+ mice. After 2 hours, mice were sacrificed and hepatocytes and nonhepatocytes were isolated. (B) LPS tissue distribution. 125I-LPS (0.125 μg/g body weight) was injected through the tail vein into SR-BI–/– and SR-BI+/+ mice. Plasma, liver, kidney, and spleen were collected after 30 minutes. LPS distribution was calculated as percentage of the total injected LPS dose. Values shown are the mean ± SD (n = 4). *P < 0.05. Similar results were found in 3 independent experiments.
Figure 10
Figure 10. Plasma from SR-BI+/+ and SR-BI–/– mice showed similar LPS neutralization activity.
Plasma (1 μl) from SR-BI+/+ and SR-BI–/– mice (pooled from 4 mice in each group) was incubated with different amounts of LPS (10 ng, 25 ng, 50 ng, and 100 ng) at 37°C for 1 hour. Endotoxin activities relative to no-plasma controls at each LPS concentration (percentage of neutralization) were determined by using a commercially available LAL assay kit. Similar results were found in 3 independent experiments.

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