An intact STAT-1 signal is required to mediate the biological activity of p17.

<p>THP-1 cells were stimulated for 18 hours with p17 (1 µg/ml) in presence or in absence of a specific STAT-1 inhibitor fludarabine (0.5 µM). At the end of treatments cellular lysates were used for Real-Time or immunoblot analysis. (A) Immunoblot of STAT-1 protein (total and phosphorylated fraction). (B) Relative mRNA expression of MCP-1, ICAM-1, PPARγ, CD40, CD80 and CD86 was expressed relative to not treated cells. Analysis was carried out in triplicate and the experiment was repeated twice. *P<0.05 versus not treated cells. #P<0.05 versus p17 stimulated cells.</p

HIV-1 p17 exerts regulatory effects on CD-14 derived PBMC isolated from HIV infected patients.

<p>CD-14 derived PBMC isolated from HIV infected patients vaccinated with an anti-p17 vaccine were stimulated ex vivo with 1 µg/ml p17 for 18 hours with or without the serum of the same patients (diluted 1∶100 in medium culture). (A–C) Activation of CD-14 derived PBMC caused by p17 (b) was reserved by p17 immune-neutralization with anti-p17 serum. Magnification 20×. (D) Relative mRNA expression of proinflammatory cytokines TNFα, IL1β, MCP-1, ICAM-1 and nuclear receptors FXR and PPARγ. (E) Relative mRNA expression of co-stimulatory molecules CD40, CD80 and CD86. (F) Relative mRNA expression of proatherogenic genes CD36 and ABCA1. Real-Time analysis was carried out in triplicate and the experiment was repeated twice. *P<0.05 versus not treated cells. #P<0.05 versus p17 stimulated cells.</p

Stimulation of THP-1 cells with p17 causes reciprocal regulation of genes involved in immune function and lipid metabolism.

<p>THP-1 cells were stimulated with 1 µg/ml p17 recombinant protein for 18 hours. (A) Relative mRNA expression of proinflammatory mediators TNFα, IL1β, MCP-1, ICAM-1 and nuclear receptors FXR and PPARγ. (B) Relative mRNA expression of co-stimulatory molecules CD40, CD80 and CD86. (C) Relative mRNA expression of proatherogenic genes CD36 and ABCA1. Real-Time analysis was carried out in triplicate and the experiment was repeated twice. *P<0.05 versus not treated cells.</p

p17 signals through RACK-1/Jak-1/STAT-1 pathway in THP-1 cells.

<p>(A) Schematic diagram showing putative interaction of p17 with syndecan-2 and the activation of RACK-1/Jak-1/STAT-1 signal transduction pathway. (B) Co-immuneprecipitation of RACK-1 with syndecan-2 and Jak-1 following stimulation of THP-1 cells with p17 recombinant protein (1 µg/ml) for 5, 15 and 30 minutes. (C) Analysis of Jak-1 protein (total and phosphorylated fraction) by immune blot in THP-1 cells stimulated with p17 (1 µg/ml) for 15, 30 and 60 minutes. (D) Analysis of STAT-1 protein (total and phosphorylated fraction) by immune blot in THP-1 cells stimulated with p17 (1 µg/ml) for 15, 30 and 60 minutes. (E) Chromatin Immunoprecipitation assay carried out in THP-1 cells left untreated or primed with 1 µg/ml p17 for 1, 6 and 18 hours. Real-Time PCR was performed on MCP-1 promoter. (F) Chromatin Immunoprecipitation assay carried out in THP-1 cells left untreated or primed with 1 µg/ml p17 for 18 hours. Real-Time PCR was performed on both MCP-1 promoter and intron-II of FXR gene. Values are normalized relative to input DNA concentration and are expressed relative to those of not treated cells immunoprecipitated with an anti IgG antibody, condition set as 1. Analysis was carried out in triplicate and the experiment was repeated twice. *P<0.05 versus not treated cells. **P<0.05 versus p17 stimulated cells.</p

HIV-1 p17 regulates pro-inflammatory, co-stimulatory and pro-atherogenic molecules in CD14-derived PBMC isolated from healthy donors.

<p>CD-14 derived PBMC isolated from healthy donors were stimulated for 18 hours with 1 µg/ml p17 recombinant protein. (A and B) Exposure to p17 drives an activated phenotype in macrophages and causes macrophages adhesion. Magnification 20×. (C) Relative mRNA expression of proinflammatory cytokines TNFα, IL1β, MCP-1, ICAM-1 and nuclear receptors FXR and PPARγ. (D) Relative mRNA expression of co-stimulatory molecules CD40, CD80 and CD86. (E) Relative mRNA expression of proatherogenic genes CD36 and ABCA1. Real-Time analysis was carried out in triplicate and the experiment was repeated twice. *P<0.05 versus not treated cells.</p

FXR agonism protects against colitis development in MyD88^−/− mice.

<p>TNBS colitis was induced in MyD88^+/+ and MyD88^−/− mice. Mice were administered 6-ECDCA, a FXR agonist, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054472#s4" target="_blank">materials and methods</a> section. (A–C) Analysis of Disease activity index (DAI) in MyD88^+/+ (A) and MyD88^−/− mice (C). (B–D) Analysis of mucosal damage score in MyD88^+/+ (B) and MyD88^−/− mice (D). (E–L) H&E staining of representative paraffin-embedded sections from distal colons after administration of vehicle (control mice), TNBS or TNBS plus 6-ECDCA in MyD88^+/+ (panels E–G) and MyD88^−/− (H–L) mice. Data are mean ± SE of 6 animals.*P<0.05 versus wild type naive mice. #P<0.05 versus wild type mice administered TNBS.</p

IRF7 binds to the an IRF7-RE located in the FXR promoter.

<p>(A) Electrophoretic Mobility shift assay (EMSA). Nuclear extracts from Raw264.7 cells left untreated or stimulated with CpG were incubated in the presence of a wild type or a mutated IRF7 biotin-labeled probe. Competition experiments were performed with a 100 fold excess of unlabeled oligo or with 1 µg IRF7 antibody. (B) Chromatin immunoprecipitation (ChIP). ChIP assay carried out in Raw264.7 cells left untreated or primed with CpG as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054472#s4" target="_blank">materials and methods</a> section. Values are normalized relative to input DNA concentration and are expressed relative to those of not treated cells immunoprecipitated with an anti IgG antibody, condition set as 1. Analysis was carried out in triplicate and the experiment was repeated twice. *P<0.05 versus not treated cells immunoprecipitated with an anti-IgG antibody. #P<0.05 versus not treated cells immunoprecipitated with an anti-IRF7 antibody.</p

FXR gene expression is regulated by TLR agonists.

<p>(A–B) Quantitative RT-PCR of FXR and TNFα genes was carried out on RNA purified from CD14 positive cells derived PBMC stimulated <i>ex vivo</i> with TLRs agonists as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054472#s4" target="_blank">materials and methods</a>. Data are mean ± SE of 3 experiments carried out in triplicate. *P<0.05 versus not treated cells. (C) Specificity of CpG effect. PCR array analysis showing the relative mRNA expression of various nuclear receptors on CD14⁺ derived PBMC stimulated <i>ex vivo</i> with TLR9 agonist CpG. (D) Quantitative RT-PCR of FXR was carried out on RNA purified from spleen-derived monocytes isolated from TLR9^+/+ and TLR9^−/− mice stimulated <i>ex vivo</i> with CpG. Data are mean of ± SE of 4 mice. *P<0.05 versus TLR9^+/+ not treated cells.</p

FXR agonism protects against colitis development in TLR9^−/− mice.

<p>TNBS colitis was induced in TLR9^+/+ and TLR9^−/− mice. Mice were administered 6-ECDCA, a FXR agonist, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054472#s4" target="_blank">materials and methods</a>. (A–C) Analysis of Disease activity index (DAI) in TLR9^+/+ (A) and TLR9^−/− mice (C). (B–D) Analysis of mucosal damage score in TLR9^+/+ (B) and TLR9^−/− mice (D). (E–L) H&E staining of representative paraffin-embedded sections from distal colons after administration of vehicle (control mice), TNBS or TNBS plus 6-ECDCA in TLR9^+/+ (panels E–G) and TLR9^−/− (H–L) mice. Data are mean ± SE of 6 animals.*P<0.05 versus wild type naive mice. #P<0.05 versus wild type mice administered TNBS.</p

A conserved IRF7 responsive element is expressed in the promoter of FXR.

<p>(A) Analysis of the FXR promoter was performed with the on-line software TFsearch. The human FXR 5'flanking region contains an IRF7-RE at −602 base pairs with respect to the transcriptional start site ATG. The murine FXR 5'flanking region contains an IRF7-RE at −787 base pairs with respect to the transcriptional start site ATG. (B) Quantitative RT-PCR of FXR and IRF7 genes was carried out on RNA purified from Raw264.7 cells stimulated with CpG. Data are mean ± SE of of 4 experiments. *P<0.05 versus not treated cells. (C) Western Blotting analysis of FXR, IRF7 and tubulin was performed on protein extracts from Raw264.7 cells stimulated with CpG. The image shown is one of three showing the same pattern. (D) Densitometric analysis of Western blot bands carried out using the Image J software. Data are the mean of three experiments. (E) Transactivation of IRF7-RE. Three copies of the murine IRF7-RE were cloned into the luciferase reporter vector pGL4. Raw264.7 cells were transiently transfected with this construct and forty-eight hours post-transfection cells were stimulated with increasing concentrations of CpG. Cellular extracts were subsequently assayed for luciferase activity. Data are the mean ± S.E. of 3 experiments carried out in triplicate. *P<0.05 versus not treated cells.</p