Abstract
High-density lipoprotein (HDL) mediates reverse cholesterol transport and is known to be protective against atherosclerosis. In addition, HDL has potent anti-inflammatory properties that may be critical for protection against other inflammatory diseases. The molecular mechanisms of how HDL can modulate inflammation, particularly in immune cells such as macrophages, remain poorly understood. Here we identify the transcriptional regulator ATF3, as an HDL-inducible target gene in macrophages that downregulates the expression of Toll-like receptor (TLR)-induced proinflammatory cytokines. The protective effects of HDL against TLR-induced inflammation were fully dependent on ATF3 in vitro and in vivo. Our findings may explain the broad anti-inflammatory and metabolic actions of HDL and provide the basis for predicting the success of new HDL-based therapies.
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Acknowledgements
We acknowledge C. Thiele (University of Bonn) for helpful discussions and J.-C. Hernandez (University of Medellin) for help with experiments. We thank C.M. De Nardo for critical reading of the manuscript. We thank T. Hai (Ohio State University) for the original Atf3-deficient mice. The work was funded by grants from US National Institutes of Health (1R01HL093262 to E.L., and 1R01HL112661 to E.L. and M.L.F.), the German Research foundation (SFB670 to E.L., SFB685 to M.Kn. and M.R.), the Excellence Cluster ImmunoSensation to E.L. and J.L.S., the Australian National Health and Medical Research Council (1006588), the Operational Infrastructure Support Program (Victoria state Government, Australia) to B.R.G.W. and D.X., and the Naito Foundation (Japan) and the Ministry of Health, Labour and Welfare and Grant-in-Aid for Scientific Research on Innovative Areas for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan to H.K. E.L. is a member of the Center for Molecular Inflammation Research at the Norwegian University of Science and Technology.
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D.D., L.I.L., H.K. and R.S. designed and performed experiments and analyzed data. S.V.S., D.X., F.A.S., J.V., A.K., M.Kr., N.B., A.G., C.L., S.Z. and N.J.H. performed experiments. S.V.S., M.B., T.U., W.K. and J.L.S. analyzed transcriptome and ChIP sequencing data. M.Kn. and M.R. provided the Atf3-deficient and matched wild-type control mice. D.L., M.L.F., B.R.G.W., P.K. and S.D.W. analyzed data and provided critical suggestions and discussions throughout the study. D.D., L.I.L., J.L.S. and E.L. designed the study and, along with S.D.W., wrote the paper.
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S.D.W. is a paid employee of CSL Behring.
Integrated supplementary information
Supplementary Figure 1 Detailed characterisation of the effect of HDL on TLR-induced cytokine secretion.
a, C57BL/6 mice were injected i.p with 2 mg recombinant HDL or PBS 6 h before subsequent injection with CpG (20 μg) and D-gal (10 mg), 1 h later serum was collected and cytokines were measured (IL-18 n=10 per group, IL-13 n=18 per group). b BMDMs were pre-treated with HDL (either 2 mg/ml or as indicated) for 6 h and stimulated overnight with CpG (100 nM or as indicated) and IL-6 measured in culture supernatants by ELISA. c, Human PBMCs were pre-treated for 6 h with HDL at indicated concentrations and stimulated overnight with LPS (2.5 ng/ml), R848 (100 ng/ml) or P3C (1 μg/ml) and IL-6 secretion measured (c, left), or PBMCs were stimulated with CpG 2336 (A-type) (1 μM) and IFNα production was measured by ELISA (c, right). a, Data are presented as mean values ±S.E.M. b, Data are presented as the mean ±S.D. and are representative of three independent experiments. c, Data is combined from three individual donors and shown as the mean ±S.E.M.
Supplementary Figure 2 HDL reduces cellular cholesterol and inhibits pro-inflammatory gene expression.
a, Immortalised-BMDMs were treated with HDL (2 mg/ml) for indicated times and cholesterol measured by mass spectrometry from cell lysates, supernatants or media with HDL. b,c, Immunoblot of BMDMs pre-treated with HDL (2 mg/ml) for 6 h and stimulated for indicated times with P3C (50 ng/ml) (b) and ELISA of IL-6 secretion (c). d, BMDMs were pre-treated with HDL or native HDL (2 mg/ml) for 6 h and stimulated with CpG (100 nM) for 4 h before mRNA expression was measured by qPCR. e, C3H/HeJ mice were injected i.p with 2 mg native HDL or control filtrate 6 h before injection with CpG (20 μg) and D-gal (10 mg), 1 h later hepatic mRNA expression was measured by qPCR (CpG n=10, native HDL+CpG n=9). f, BMDMs were pre-treated for 6 h with HDL before 4 h with CpG (100 nM) and Actinomycin D (5 μg/ml) for the indicated times to asses the half-life of IL-6 transcripts. Data is normalised to 0 min Actinomycin D sample for respective conditions. g, BMDMs were pre-treated for 12 h HDL before CpG for 4 h and cyclohexamide (10 μg/ml) treatment for the indicated times to assess the half life of IL-1β protein (relative to β-actin). a, A representative graph of two individual experiments is presented (mean ±S.D.). b-d, A representative blot (b) and ELISA (mean ±S.D.) (c,d) of three individual experiments is shown. e, Data are presented as mean values ±S.E.M, CpG versus native HDL+CpG *p<0.05, **p<0.01. f, A representative graph from two independent experiments is shown. g, A single immunoblot is shown and densitometric analysis of IL-1β combined from three independent experiments (mean ±S.E.M).
Supplementary Figure 3 Transcriptome analysis of BMDMs treated with HDL.
a-c,e,f, Transcriptome data are derived from BMDMs pre-treated for 6 h with HDL (2 mg/ml) then stimulated for 4 h with CpG (100 nM). a, Principal component analysis of all genes demonstrating sample relationships and group associations of individual samples. b, Hierarchical clustering of the 1000 most variable genes within the dataset. c Visualisation of genes (fold change values) involved in the cholesterol biosynthesis pathway from HDL treated BMDMs. d, Immortalised-BMDMs were treated with 2 mg/ml HDL for indicated times and cholesterol precursors were measured by mass spectrometry. e, Network visualization of Gene Ontology Enrichment Analysis (GOEA) based on transcripts reduced by CpG and counter-regulated by HDL (red nodes: GO-terms, red edges: GO-term relations) or induced by CpG and counter-regulated by HDL (blue edges and nodes). f, Workflow scheme of transcription factor prediction modelling. a-c,e,f, At least three biological replicates per condition were generated. d, Representative graphs of two individual experiments are presented (mean ±S.D.).
Supplementary Figure 4 ATF3 does not bind the promoters of control genes but is induced by HDL in a model of atherosclerosis.
a, Genomic loci of Il18 and Il13 with ChIP-Seq signals for ATF3 binding under the various stimulation conditions. b, qPCR analysis of ATF3 mRNA expression in Kupffer cells or hepatocytes isolated from Apoe-deficient mice fed on a Western diet and injected i.v. with PBS or HDL (100 mg/kg) (n=5 per group). a, Data was obtained from 3 biological replicates. b, Data are shown as the mean ±S.E.M, PBS versus HDL injected mice **p<0.01.
Supplementary Figure 5 Transcriptome analysis of WT vs Atf3-deficient BMDMs.
Microarray analysis of WT or Atf3-deficient BMDMs pre-treated with 2 mg/ml HDL for 6 h and subsequently stimulated with CpG (100 nM) or P3C (50 ng/ml) for 4 h. a, Visualisation of transcripts induced or repressed by P3C in WT, counter regulated by HDL pre-treatment, and no longer modified in Atf3-deficient BMDMs. b, Venn diagrams show the overlap between CpG and P3C conditions from genes identified using the model described. c Network visualization of GOEA: GO-terms (nodes) and their relation (edges) based on ATF3-independent genes are shown in blue, and those based on ATF3-dependent genes are shown in red. d, Visualisation in fold change of genes involved in the cholesterol biosynthesis pathway from HDL treated WT or Atf3-deficient BMDMs. e, 27 of the 130 transcripts altered by CpG, counter-regulated by HDL and no longer modified in Atf3-deficient BMDMs are direct ATF3-target genes. Network visualization of GOEA based on transcripts reduced by CpG, counter-regulated by HDL and no longer modified in Atf3-deficient BMDMs showing ATF3 binding by ChIP-Seq (red nodes: GO-terms, red edges: GO-term relations) or induced by CpG and counter-regulated by HDL and no longer modified in Atf3-deficient BMDMs showing ATF3 binding (blue edges and nodes). a-e, At least three biological replicates per condition were generated and analysed.
Supplementary Figure 6 Transcriptome analysis of carotid injury model.
Transcriptome data derived from RNA of carotid arteries of mice subjected to endothelial injury 3 h prior to HDL (20 ug/kg) or PBS i.v. injection. a Hierarchical clustering of the 2073 most variable genes within the dataset. b, Network visualisation of Gene ontology enrichment analysis (GOEA) based on transcripts regulated in macrophages in vitro and in carotid arteries in vivo. Enrichment scores of GO-terms based on genes upregulated or downregulated by HDL in vivo. a, b, At least three biological replicates per condition were generated and analysed.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–6 (PDF 2210 kb)
Transcription factor binding prediction identifies ATF3.
Microarray analysis of BMDMs pre-treated for 6 h with HDL (2 mg/ml) then stimulated with CpG (100 nM) for 4 h. The most significantly repressed genes (Fold change <-3; False discovery rate p<0.05) following CpG stimulation in the presence of HDL (33 input genes) were subjected to transcription factor binding prediction as described in the Methods. (XLSX 60 kb)
ATF3 is required for a large number of HDL mediated effects on TLR-driven changes in gene expression.
a-f Microarray analysis of WT and Atf3-deficient BMDMs pre-treated with HDL (2 mg/ml) for 6 h and subsequently stimulated for 4 h with CpG (100 nM) or P3C (50 ng/ml). Genes modulated in WT BMDMs following TLR stimulation and counter regulated by HDL pre-treatment that were no longer regulated by HDL in Atf3-deficient BMDMs; a all genes (n=224), b genes induced by CpG but repressed by HDL (n=93), c genes repressed by CpG but induced by HDL (n=53), d genes induced by P3C but repressed by HDL (n=118) and, e genes repressed by P3C but induced by HDL (n=58). f Annotation of supplemental tables 2a-e. (XLSX 157 kb)
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De Nardo, D., Labzin, L., Kono, H. et al. High-density lipoprotein mediates anti-inflammatory reprogramming of macrophages via the transcriptional regulator ATF3. Nat Immunol 15, 152–160 (2014). https://doi.org/10.1038/ni.2784
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DOI: https://doi.org/10.1038/ni.2784
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