Volume 12, Issue 4, Pages 805-816 (October 2003) Crosstalk between LXR and Toll-like Receptor Signaling Mediates Bacterial and Viral Antagonism of Cholesterol Metabolism  Antonio Castrillo, Sean B Joseph, Sagar A Vaidya, Margaret Haberland, Alan M Fogelman, Genhong Cheng, Peter Tontonoz  Molecular Cell  Volume 12, Issue 4, Pages 805-816 (October 2003) DOI: 10.1016/S1097-2765(03)00384-8

Figure 1 Bacterial and Viral Infections Inhibit LXR-Dependent Gene Expression Thioglycolate-elicited peritoneal macrophages from C57BL/6 mice were infected in vitro with either E. coli (A and B) or influenza A (C and D). Simultaneous with infection, cells were treated with vehicle (DMSO), 1 μM GW3965, or 1 μM T1317 for 48 hr. Gene expression was analyzed by Northern blotting (A and C) or real-time quantitative PCR (B and D). 36B4 was used as a control for loading and integrity of the RNA. Molecular Cell 2003 12, 805-816DOI: (10.1016/S1097-2765(03)00384-8)

Figure 2 Ligands for TLR3 and TLR4 Selectively Inhibit the Expression of LXR Target Genes (A) Lipid A but not TNFα or IL-1β inhibits ABCA1 expression in response to LXR ligands. Peritoneal macrophages from C57BL/6 mice were cultured in the presence of vehicle or 50 ng/ml lipid A, 50 ng/ml TNFα, 50 ng/ml IL-1β, and/or 1 μM GW3965 or T1317 for 24 hr. ABCA1 expression was measured by real-time quantitative PCR. (B) Inhibition of LXR-dependent gene expression by inflammatory signals does not correlate with cytokine induction. Macrophages were treated with vehicle or the indicated concentration (ng/ml) of lipid A, TNFα, or IL-1β, and/or 1 μM GW3965 for 24 hr. (C) Ligands for TLR3 and TLR4 but not TLR2 or TLR9 inhibit the expression of LXR target genes. Macrophages were treated with vehicle, 5 μg/ml LTA, 50 ng/ml lipid A, 5 μg/ml poly I:C, or 100 nM CpG in the presence or absence of 1 μM GW3965 for 24 hr. (D) LXR agonist does not the block induction of TLR3-responsive genes. RAW264.7 macrophages were treated with vehicle or 5 μg/ml poly I:C in the presence or absence of 1 μM GW3965 for 24 hr. (E) TLR ligands inhibit the expression of LXR but not PPARγ or PPARδ target genes. Peritoneal macrophages were treated with vehicle or 50 ng/ml lipid A or 5 μg/ml poly I:C, in the presence or absence of 1 μM GW3965, 1 μM GW7845 (PPARγ ligand), or 1 μM GW1516 (PPARδ ligand) for 24 hr. Gene expression was measured by real-time quantitative PCR (A and E) or Northern blotting (B, C, and D). Molecular Cell 2003 12, 805-816DOI: (10.1016/S1097-2765(03)00384-8)

Figure 3 TLR3/4 Ligands Inhibit ABCA1 Protein Expression and Macrophage Cholesterol Efflux (A) Lipid A inhibits ABCA1 protein expression in murine macrophages. Thioglycolate-elicited peritoneal macrophages were treated with vehicle or 50 ng/ml lipid A in the presence or absence of 1 μM GW3965 for 48 hr. Protein expression was determined by Western blotting with anti-ABCA1 antibody. Lysate from mock or ABCA1-transfected HEK-293 cells served as a control for antibody specificity. N.S. indicates nonspecific protein. (B) Ligands for TLR3/4 inhibit cholesterol efflux. Peritoneal macrophages from C57BL/6 mice were loaded with 3H-cholesterol and treated with vehicle or 50 ng/ml lipid A, 5 μg/ml poly I:C, 5 μg/ml LTA, or 50 ng/ml TNFα in the presence or absence of 1 μM GW3965. Data are presented as apoAI-dependent cholesterol efflux. (*) P < 0.05; (**) P < 0.01 by student's t test. Molecular Cell 2003 12, 805-816DOI: (10.1016/S1097-2765(03)00384-8)

Figure 4 TLR3/4 Ligands Inhibit LXR Activity on Its Target Promoters (A) Repression of ABCA1 promoter expression by TLR3/4 ligands is mediated by the LXRE. RAW 264.7 cells were transiently transfected with ABCA1-luciferase or ABCA1-LXREmut-luciferase reporters and expression vectors for LXRα and RXRα. After transfection, cells were treated with 50 ng/ml lipid A and/or 1 μM GW3965. Data are presented as normalized luciferase activity. (B) TLR ligands inhibit LXR activation of an isolated LXRE reporter. Cells were transfected as in (A) except that an LXRE-luciferase reporter was used. (C) TLR ligands do not antagonize PPARγ-dependent transcription. RAW 264.7 cells were transfected with a PPRE-luciferase reporter and expression vectors for PPARγ and RXRα. After transfection, cells were treated with 50 ng/ml lipid A and/or 1 μM of the PPARγ agonist GW7845. (D) Expression of TLRs in HEK-293 cells facilitates activation of an NF-κB reporter by TLR ligands. HEK-293 cells were transiently transfected with an NF-κB-luciferase reporter and expression vectors for LXRα/RXRα and/or expression vectors for TLR2, TLR3, or TLR4 and MD-2. After transfection, cells were treated with 5 μg/ml LTA, 5 μg/ml poly I:C, or 50 ng/ml lipid A and/or 1 μM GW3965. (E) Expression of TLR3 or TLR4 facilitates repression of the ABCA1 promoter by TLR ligands. HEK-293 cells were transfected as in (D) except that ABCA1-luciferase was used as the reporter. Molecular Cell 2003 12, 805-816DOI: (10.1016/S1097-2765(03)00384-8)

Figure 5 Inhibition of LXR by TLR3/4 Ligands Is Independent of MyD88 and NF-κB and Does Not Require Production of Type I Interferons (A) Schematic of the MyD88-dependent and alternative TLR3/4 signaling pathways. (B) The ability of TLR ligands to inhibit ABCA1 expression is preserved in MyD88 null macrophages. Peritoneal macrophages derived from wild-type or MyD88 null mice were treated with vehicle, 5 μg/ml LTA, 50 ng/ml lipid A, 5 μg/ml poly I:C, or 100 nM CpG in the presence or absence of 1 μM GW3965. (C) The ability of TLR ligands to inhibit macrophage cholesterol efflux does not require MyD88. Peritoneal macrophages derived from wild-type or MyD88 null mice were loaded with 3H-cholesterol and treated with TLR and/or LXR ligands as in (B). Data are presented as apoAI-dependent efflux. (D) Inhibition of LXR-dependent gene expression by TLR3/4 does not require NF-κB. RAW-vector and RAW-IκB-DA cells were treated with TLR and/or LXR ligands as in (B). (E and F) Repression of ABCA1 apoE expression by TLR3/4 ligands is preserved in IFNAR null macrophages. Macrophages derived from wild-type or IFNAR null mice were treated with TLR and/or LXR ligands. Gene expression was determined by real-time quantitative PCR (B, E, and F) or Northern blotting (D). Molecular Cell 2003 12, 805-816DOI: (10.1016/S1097-2765(03)00384-8)

Figure 6 Crosstalk between TLR and LXR Signaling Is Mediated by IRF3. (A) Expression of IRF3 blocks LXR-dependent activation of the ABCA1 promoter. RAW264.7 cells were transiently transfected with ABCA1-luciferase reporter and expression vectors for LXRα/RXRα, IRF3, or IRF3-DN as indicated. After transfection, cells were treated with 5 μg/ml poly I:C and/or 1 μM GW3965. (B) Stable expression of IRF3 in RAW 264.7 cells inhibits endogenous ABCA1 expression. RAW-vector, RAW-IRF3, or RAW-IRF3-DN macrophages were treated with 50 ng/ml lipid A, 5 μg/ml poly I:C, and/or 1 μM GW3965. (C) Expression of a dominant-negative IRF3 renders the LXR pathway insensitive to inhibition by TLR ligands. RAW-vector or RAW-IRF3-DN cells were treated with TLR and/or LXR ligands as above. (D) The ability TLR ligands to antagonize ABCA1 expression is abolished in IRF3 null macrophages. Peritoneal macrophages derived from wild-type or IRF3 null mice were treated with TLR and/or LXR ligands as above. Gene expression was measured by real-time quantitative PCR (B and D) or Northern blotting (C). (E) IRF3 is required for TLR-dependent inhibition of cholesterol efflux. Macrophages derived from wild-type or IRF3 null mice were loaded with 3H-cholesterol and treated with TLR and/or LXR ligands. Cholesterol efflux to apoAI was determined as above. Data are presented as apoAI-dependent efflux. (*) P < 0.05. Molecular Cell 2003 12, 805-816DOI: (10.1016/S1097-2765(03)00384-8)

Figure 7 Expression of p300 Stimulates LXR-Dependent Gene Expression and Alleviates Repression by the TLR3-IRF3 Pathway (A) Activation of IRF3 by the kinase TBK-1 potentiates inhibition of LXR. RAW264.7 cells were transiently transfected with ABCA1-luciferase reporter and expression vectors for LXRα/RXRα, and/or IRF3 and TBK-1 as indicated. After transfection, cells were treated with 5 μg/ml poly I:C and/or 1 μM GW3965. (B) Expression and activation of LXR does not block TLR3-dependent induction of the IFNβ promoter. RAW264.7 cells were transfected and treated with ligands as in (A). (C) LXRα does not interact directly with IRF3 in vitro. Bacterially expressed GST-LXRα fusion protein was used in a pull down assay with 35S-labeled in vitro translated IRF3, IRF3-DN, or RXRα. (D) Overexpression of p300 blocks the ability of TLR3 and IRF3 to inhibit LXR transcriptional activity. RAW264.7 cells were transiently transfected with ABCA1-luciferase reporter and pCMX-based expression vectors for LXRα/RXRα, IRF3, and/or p300 as indicated. After transfection, cells were treated with 5 μg/ml poly I:C and/or 1 μM GW3965. Molecular Cell 2003 12, 805-816DOI: (10.1016/S1097-2765(03)00384-8)

Figure 8 Activation of TLR3 Inhibits LXR Target Gene Expression In Vivo 10-week-old male C57BL/6 mice (5 per group) were gavaged for 3 days with GW3965 (20 mg/kg per day) or vehicle (0.5% methylcellulose). Mice also received an intraperitoneal injection of 50 μg poly I:C or saline control. All mice received their final dose of GW3965/vehicle and/or poly I:C/saline 2 hr before tissue collection after a 12 hr fast. Gene expression was determined by real-time quantitative PCR individually for each animal and the average for each group is presented. Data were analyzed using the student's t test. (*) P < 0.05. Molecular Cell 2003 12, 805-816DOI: (10.1016/S1097-2765(03)00384-8)