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. 2016 Apr 1;196(7):3135-47.
doi: 10.4049/jimmunol.1501709. Epub 2016 Mar 2.

Human SR-BI and SR-BII Potentiate Lipopolysaccharide-Induced Inflammation and Acute Liver and Kidney Injury in Mice

Irina N Baranova  1 , Ana C P Souza  2 , Alexander V Bocharov  3 , Tatyana G Vishnyakova  1 , Xuzhen Hu  2 , Boris L Vaisman  4 , Marcelo J Amar  4 , Zhigang Chen  1 , Yana Kost  1 , Alan T Remaley  4 , Amy P Patterson  5 , Peter S T Yuen  2 , Robert A Star  2 , Thomas L Eggerman  6
Affiliations

Human SR-BI and SR-BII Potentiate Lipopolysaccharide-Induced Inflammation and Acute Liver and Kidney Injury in Mice

Irina N Baranova et al. J Immunol. .

Abstract

The class B scavenger receptors BI (SR-BI) and BII (SR-BII) are high-density lipoprotein receptors that recognize various pathogens, including bacteria and their products. It has been reported that SR-BI/II null mice are more sensitive than normal mice to endotoxin-induced inflammation and sepsis. Because the SR-BI/II knockout model demonstrates multiple immune and metabolic disorders, we investigated the role of each receptor in the LPS-induced inflammatory response and tissue damage using transgenic mice with pLiv-11-directed expression of human SR-BI (hSR-BI) or human SR-BII (hSR-BII). At 6 h after i.p. LPS injection, transgenic hSR-BI and hSR-BII mice demonstrated markedly higher serum levels of proinflammatory cytokines and 2- to 3-fold increased expression levels of inflammatory mediators in the liver and kidney, compared with wild-type (WT) mice. LPS-stimulated inducible NO synthase expression was 3- to 6-fold higher in the liver and kidney of both transgenic strains, although serum NO levels were similar in all mice. Despite the lower high-density lipoprotein plasma levels, both transgenic strains responded to LPS by a 5-fold increase of plasma corticosterone levels, which were only moderately lower than in WT animals. LPS treatment resulted in MAPK activation in tissues of all mice; however, the strongest response was detected for hepatic extracellular signal-regulated protein kinase 1 and 2 and kidney JNK of both transgenic mice. Histological examination of hepatic and renal tissue from LPS-challenged mice revealed more injury in hSR-BII, but not hSR-BI, transgenic mice versus WT controls. Our findings demonstrate that hSR-BII, and to a lesser extent hSR-BI, significantly increase LPS-induced inflammation and contribute to LPS-induced tissue injury in the liver and kidney, two major organs susceptible to LPS toxicity.

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Figures

Figure 1
Figure 1. Western blot analyses of liver and kidney tissue samples
Western blot analyses for hSR-BI and hSR-BII protein expression were performed in liver and kidney tissue samples from WT (lanes 1, 4), hSR-BI tgn (lanes 2, 5) and hSR-BII tgn (lanes 3, 6) mice. hSR-BI and hSR-BII protein expression was detected by using either anti-human SR-BI and anti-human SR-BII custom antibodies (against specific peptides from C-terminal domains) or anti-human SR-BI/BII antibody (against 104–294 amino acid peptide, BD Biosciences). mSR-BI expression was detected by anti-SR-BI antibody (against C-terminal 450–509 amino acid peptide, Novus Biologicals, cat. # NB400-101). Protein expression of β-actin was measured as the loading control. ImageJ software was used to quantify the protein bands intensity, and the relative optical density (ROD) of SR-BI and SR-BII specific bands was calculated versus corresponding β-actin values (upper panel).
Figure 2
Figure 2. Plasma levels of inflammatory cytokines in WT, hSR-BI tgn and hSR-BII tgn mice challenged with LPS
LPS (1 mg/kg, IP) or PBS was injected into WT, hSR-BI and hSR-BII tgn mice. Six hours after the LPS injection, mice were euthanized for plasma and organ collection. Plasma levels of IL-6 (A), MIP-2 (B), CXCL1 (C) and IL-1β (D) were determined by ELISA. Values are the mean ± SD (n=5). ** P<0.01, *** p<0.005, vs WT LPS-treated levels.
Figure 3
Figure 3. Hepatic gene expression of inflammatory cytokines in WT, hSR-BI and hSR-BII transgenic mice challenged with LPS
LPS (1 mg/kg, IP) or PBS was injected into WT, hSR-BI and hSR-BII tgn mice. Six hours after the LPS injection, mice were euthanized and liver tissue was collected for mRNA extraction and qRT-PCR as described in Materials and Methods. Expression levels of IL-6 (A), CXCL1 (B), IL-1β (C) and TNF-α (D) were normalized by GAPDH and presented as the fold change relative to PBS-treated control. Values shown are the mean ± SD (n=3, for PBS-treated groups, n=5 for LPS-treated groups).* P<0.05, ** P<0.01 vs WT LPS-treated mice.
Figure 4
Figure 4. Kidney gene expression of inflammatory cytokines in WT, hSR-BI and hSR-BII transgenic mice challenged with LPS
LPS (1 mg/kg, IP) or PBS was injected into WT, hSR-BI and hSR-BII tgn mice. Mice were euthanized after 6 hours; kidney samples were collected and used for mRNA extraction and qRT-PCR as described in Materials and Methods. Expression levels of IL-6 (A), CXCL1 (B), IL-1β (C) and TNF-α (D) were normalized by GAPDH and presented as the fold change relative to PBS-treated control. Values shown are the mean ± SD (n=3, for non-treated groups, n=5 for LPS-treated groups). * P<0.05, ** P<0.01 vs WT LPS-treated mice.
Figure 5
Figure 5. NLRP3 mRNA expression in the liver and kidney of WT, hSR-BI and hSR-BII transgenic mice challenged with LPS
Mice were euthanized and liver and kidney samples were collected for mRNA extraction and qRT-PCR as described in Materials and Methods. Expression of liver (A) and kidney (B) NLRP3 was normalized by GAPDH and presented as the fold change relative to PBS-treated control. Values shown are the mean ± SD (n=3, for PBS-treated groups, n=5 for LPS-treated groups. *** P<0.001 vs WT LPS-treated mice.
Figure 6
Figure 6. Liver and kidney expression of iNOS and plasma nitrite/nitrate (NOx) and corticosterone levels in LPS-challenged mice
Liver (A) and kidney (B) mRNA expression of iNOS was quantified as we previously described in figure legends 3 and 4. C. Plasma NOx levels of WT, hSR-BI and hSR-BII transgenic mice were measured 6 hours following LPS injection using a colorimetric kit. Values are the mean ± SD (n=5). D. The corticosterone concentration was quantified by ELISA using plasma from hSR-BI and hSR-BII tgn mice and WT littermates 6 hours following LPS injection (n=10 for control PBS-treated groups, and n=5 for LPS-treated group, with duplicate measurements). ** P<0.01 vs WT LPS-treated mice.
Figure 7
Figure 7. LPS-induced histological liver damage in various mice
A. Semi-quantitative histological analysis of liver injury. Liver injury was defined as the amount of destruction of hepatic lobules, infiltration of inflammatory cells, hemorrhage, and hepatocyte necrosis, and scored from 1 through 4 according to % area of involvement per HPF (400×). Liver damage scores are presented for mice that received PBS (n=4–7/group, open bars) or an LPS injection (n=5/group, dashed bars). B. Representative images (400×) of liver sections stained by PAS from each group (mice that received PBS: WT – image B1, hSR-BI tgn – image B2, and hSR-BII tgn – image B3), mice that received LPS: WT – image B4, hSR-BI tgn – image B5, and hSR-BII tgn – image B6). The description of the arrows is in the Results section.
Figure 8
Figure 8. LPS-induced kidney damage in various mice
Semi-quantitative analysis of kidney injury. Kidney tubular damage was defined as tubular epithelial swelling, loss of brush border, vacuolar degeneration, necrotic tubules, cast formation, and desquamation, and scored from 1 through 4 according to % area of involvement per HPF (400×). A. Tubular damage scores of mice that received PBS (N=4–5/group, open bars), and mice that received an LPS injection (N=5/group, dashed bars). B. Representative images (400×) of kidney sections stained by PAS from each group (mice that received PBS: WT – image B1, hSR-BI tgn – image B2, and hSR-BII tgn – image B3), mice that received. LPS: WT – image B4, hSR-BI tgn – image B5, and hSR-BII tgn – image B6. The description of the arrows is in the Results section.
Figure 9
Figure 9. Western Blot analyses of MAPKs activity in the liver and kidney of LPS-treated WT and SR-B transgenic mice
Four hours after PBS or LPS (1 mg/kg, IP) injection into WT, hSR-BI and hSR-BII tgn mice (n=3 for each group), mice were sacrificed; organs were collected and processed for assessment of MAPKs phosphorylation as described in the Materials and Methods section. A. MAPK activity of liver samples using antibodies against phospho-ERK1/2 (upper panel) or phospho-p38 (bottom panel). B. MAPK activity of kidney samples using antibodies against phospho-ERK1/2 (top panel), phospho-JNK (middle panel), or phospho-p38 (bottom panel). Equal loading of samples was ensured by using anti-ERK1/2, anti-JNK or anti-p38 MAPK antibodies against total (non-phosphorylated) MAPK protein.
Figure 10
Figure 10. Dose-dependent LPS-induced IL-6 secretion in primary kidney epithelial cells (KECs) from WT, hSR-BI and hSR-BII transgenic mice
A. KECs were incubated with LPS (0, 10, 50, 100 and 500 ng/ml) in serum-free DMEM containing 2mg/ml BSA and 10mM HEPES pH 7.4 for 18 hours. Media were collected and assayed for IL-6 secretion by ELISA. * P<0.05, ** P<0.01 vs WT LPS-treated cells. B. Western blot analysis of KECs from WT (lane 1), hSR-BI tgn (lane 2) and hSR-BII tgn (lane 3) mice. The expression of hSR-BI and mSR-BI expression was detected utilizing an anti-SR-BI antibody (against C-terminal 450–509 amino acid peptide, Novus Biologicals, cat. # NB400-101) and hSR-BI/hSR-BII protein was detected using anti-human SR-BI/BII loop antibody (against 104–294 amino acid peptide, BD Biosciences). Protein expression of β-actin was measured as the loading control.
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