Deletion of scavenger receptor A gene in mice resulted in protection from septic shock and modulation of TLR4 signaling in isolated peritoneal macrophages

Abstract

Scavenger receptor A (Sra), also known as macrophage scavenger receptor 1 (Msr1), is a surface glycoprotein preferentially present in macrophages that plays a primary role in innate immunity. Previous studies have shown that Sra is a modifier gene for the response to bacterial LPS in mice at the level of IL-10 production, in particular. In the present study, we found that Sra(-/-) mice are more resistant to septic shock induced by cecal ligation and puncture than wild-type C57BL/6 J (B6) mice. In addition, Sra(-/-) mice displayed initial elevated high density lipoprotein (HDL) circulating levels. Naïve peritoneal macrophages (PMs) were isolated from Sra(-/-) mice to understand the possible protective mechanism. Incubation of these cells with LPS was found to modulate TLR4 signaling, leading to a reduction in IL-10 and IL-6 mRNA levels, but not TNF-α expression, at low concentrations of LPS in comparison with PMs isolated from B6 mice. No differences were found in LPS binding between PMs derived from Sra(-/-) or B6 mice. The lack of Sra binding to LPS was confirmed after transfection of Chinese hamster ovary (CHO) cells with the Sra gene. The contribution of Sra to the outcome of sepsis may be a combination of changes in TLR4 signaling pathway and elevated levels of HDL in circulation, but also LPS toxicity.

Figure 1

Survival of Sra(−/−) and B6 mice after CLP. Male Sra(−/−) or B6 mice were NPO for 16 h before the procedure. Anesthetized mice were subjected to CLP (1.5-cm ligation) and puncture with 18 - or 16-guage needle. Survival was monitored continuously for 120 h (18-gauage) or 72 h (16-guage) after CLP. Statistical significance was analyzed by log-rank test. (A) CLP (18-guage), Sra(−/−) n=10, B6 n=20, *P<0.05 Sra(−/−) vs. B6. (B) CLP (16-guage), Sra(−/−) n=20, B6 n=20, *P<0.05 Sra(−/−) vs. B6.

Figure 2

LPS-induced cytokine profile in PMϕs isolated from Sra(−/−) or B6 mice. Naïve PMϕs were isolated from Sra(−/−) or B6 mice by peritoneal lavage in the absence of recruiting agents, as described in the ‘Materials and methods’. Cells were incubated (or not) with LPS (1 ng/ml or 100 ng/ml as indicated) for 3 h at 37°C in RPMI 1640 medium containing FBS (10%). At the end of the incubation time, RNA was then isolated, DNase treated, and reverse-transcribed to cDNA. TNF-α, IL-10, and IL-6 mRNA levels were determined by qRT-PCR using the standard curve method. The housekeeping gene GAPDH was used to normalize the data. Results are expressed as average±SE (n=4–5). Significance was analyzed by student’s t-test between cells under each condition isolated from Sra(−/−) or B6 mice. P<0.02 for IL-10 Sra(−/−) vs. B6. P<0.02 for IL-6 Sra(−/−) vs. B6.

Figure 3

Comparison of CD14 and TLR4 mRNA, and surface levels on Sra(−/−) and B6-derived PMϕs before and after incubation with LPS. Naïve PMϕs were isolated from Sra(−/−) or B6 mice by peritoneal lavage in the absence of recruiting agents, as described in the ‘Materials and methods’. (A) PMϕs were stained with a combination of FITC-conjugated anti-CD14 and APC-conjugated anti-TLR4/MD2 Abs. Fluorescence was acquired using a BD FACSCanto II flow cytometer and analyzed by FlowJo software. (B) Cells were incubated (or not) with LPS (1 ng/ml) for 3 h at 37°C in RPMI 1640 medium containing FBS (10%). RNA was then isolated, DNase treated, and reverse-transcribed to cDNA. CD14 and TLR4 mRNA levels were determined by qRT-PCR using the standard curve method. The housekeeping gene GAPDH was used to normalize the data. Results are expressed as average±SE (n=4–5). Significance was analyzed by student’s t-test between cells under each condition isolated from Sra(−/−) or B6 mice. (C) LPS-treated PMϕs were stained with a combination of FITC-conjugated anti-CD14 and APC-conjugated anti-TLR4/MD2 Abs. Fluorescence was acquired using a BD FACSCanto II flow cytometer and analyzed by FlowJo software. Representative histogram plots are shown with filled grey histograms for control PMϕs (untreated) and open black histograms for LPS-treated PMϕs. Dashed histograms represent CD14 or TLR4 corresponding isotype control Abs.

Figure 4

PMϕs isolated from Sra(−/−) or B6 mice showed an identical LPS binding profile. Naïve PMϕs were isolated from Sra(−/−) or B6 mice by peritoneal lavage in the absence of recruiting agents, as described in the ‘Materials and methods’. (A) Isolated PMϕs were characterized by staining with APC-conjugated anti-F4/80 Abs and biotin-conjugated LPS followed by AlexaFluor488-conjugated streptavidin. An isotype control was also included. Notice that there is no difference in LPS binding between PMϕs isolated from Sra(−/−) or B6 mice. Fluorescence was acquired using a BD FACSCanto II flow cytometer and analyzed by FlowJo software.

Figure 5

CHO cells transfected with Sra did not display any LPS binding. CHO cells were transfected with murine Sra (plasmid) using Fugene HD. After 24 h of the transfection, cells were analyzed using various reagents. (A) Mock-transfected CHO cell; (B–E) Sra transfected CHO cells. Cells were stained with biotin-conjugated LPS followed by AlexaFluor594-conjugated streptavidin (A, B), anti-Sra Ab and AlexaFluor488-conjugated anti-rabbit secondary Ab (C), AlexaFluor488-conjugated anti-rabbit secondary Ab in absence of primary Ab (D), and AlexaFluor488-conjugated-Ac-LDL (E). Notice the lack of LPS binding to Sra transfected cells.

Figure 6

CHO cells transfected with SraI, SraII, or both did not bind LPS. CHO cells were transfected with murine SraI, SraII, or both (plasmid) using Fugene HD. After 24 h of the transfection, cells were analyzed using various reagents. SraI-transfected CHO cells (A–D), SraII (E–H), SraI and SraII (I–L). Cells were stained with anti-Sra Ab and AlexaFluor488-conjugated anti-rabbit secondary Ab (A, C, E, I); AlexaFluor488-conjugated anti-rabbit secondary Ab in absence of primary Ab (B, F, J); AlexaFluor488-conjugated-Ac-LDL (C, G, K); biotin-conjugated LPS followed by AlexaFluor594-conjugated streptavidin (D, H, L).

Figure 7

Lipid profile of Sra(−/−) and B6 mice. Male Sra(−/−) (n=10) or B6 (n=12) mice were NPO for 16 h before the procedure. Anesthetized mice were subjected to CLP (1.5-cm ligation) and puncture with a 16-guage needle or were non-operated [NO; B6 n=5; Sra(−/−) n=3]. The lipid profile was determined in plasma samples from NO mice, 6 or 20 h after CLP. Total cholesterol (A), HDL (B) calculated LDL (C), triglyceride levels (D), calculated VLDL (E), and lipase (F). Significance was analyzed using a one-way ANOVA followed by Newman-Keuls multiple comparison test for each time point (P<0.0001).