High-density lipoprotein mediates anti-inflammatory reprogramming of macrophages via the transcriptional regulator ATF3

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: HDL inhibits TLR-induced cytokine production from macrophages in vivo and in vitro.
Figure 2: HDL inhibits TLR-induced proinflammatory cytokine transcription.
Figure 3: Microarray analysis identifies Atf3 as a candidate gene for the anti-inflammatory function of HDL.
Figure 4: HDL induces ATF3 expression.
Figure 5: ATF3 is active after induction by HDL.
Figure 6: ATF3 mediates much of the transcriptional response to HDL treatment of macrophages.
Figure 7: ATF3 is required for the anti-inflammatory effect of HDL in vitro and in vivo.

Accession codes

Primary accessions

Gene Expression Omnibus

References

  1. 1

    Takeuchi, O. & Akira, S. Pattern recognition receptors and inflammation. Cell 140, 805–820 (2010).

  2. 2

    Medzhitov, R. & Horng, T. Transcriptional control of the inflammatory response. Nat. Rev. Immunol. 9, 692–703 (2009).

  3. 3

    Gilchrist, M. et al. Systems biology approaches identify ATF3 as a negative regulator of Toll-like receptor 4. Nature 441, 173–178 (2006).

  4. 4

    Hai, T., Wolford, C.C. & Chang, Y.S. ATF3, a hub of the cellular adaptive-response network, in the pathogenesis of diseases: is modulation of inflammation a unifying component? Gene Expr. 15, 1–11 (2010).

  5. 5

    Whitmore, M.M. et al. Negative regulation of TLR-signaling pathways by activating transcription factor-3. J. Immunol. 179, 3622–3630 (2007).

  6. 6

    Duewell, P. et al. NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals. Nature 464, 1357–1361 (2010).

  7. 7

    Seneviratne, A.N., Sivagurunathan, B. & Monaco, C. Toll-like receptors and macrophage activation in atherosclerosis. Clin. Chim. Acta 413, 3–14 (2012).

  8. 8

    Gordon, T., Castelli, W.P., Hjortland, M.C., Kannel, W.B. & Dawber, T.R. High density lipoprotein as a protective factor against coronary heart disease. The Framingham Study. Am. J. Med. 62, 707–714 (1977).

  9. 9

    Mineo, C. & Shaul, P.W. Novel biological functions of high-density lipoprotein cholesterol. Circ. Res. 111, 1079–1090 (2012).

  10. 10

    Schwartz, G.G. et al. Effects of dalcetrapib in patients with a recent acute coronary syndrome. N. Engl. J. Med. 367, 2089–2099 (2012).

  11. 11

    Boden, W.E. et al. Niacin in patients with low HDL cholesterol levels receiving intensive statin therapy. N. Engl. J. Med. 365, 2255–2267 (2011).

  12. 12

    Rader, D.J. & Tall, A.R. The not-so-simple HDL story: Is it time to revise the HDL cholesterol hypothesis? Nat. Med. 18, 1344–1346 (2012).

  13. 13

    Khera, A.V. et al. Cholesterol efflux capacity, high-density lipoprotein function, and atherosclerosis. N. Engl. J. Med. 364, 127–135 (2011).

  14. 14

    Libby, P., Ridker, P.M. & Hansson, G.K. Progress and challenges in translating the biology of atherosclerosis. Nature 473, 317–325 (2011).

  15. 15

    Murphy, A.J., Westerterp, M., Yvan-Charvet, L. & Tall, A.R. Anti-atherogenic mechanisms of high density lipoprotein: effects on myeloid cells. Biochim. Biophys. Acta 1821, 513–521 (2012).

  16. 16

    Norata, G.D., Pirillo, A., Ammirati, E. & Catapano, A.L. Emerging role of high density lipoproteins as a player in the immune system. Atherosclerosis 220, 11–21 (2012).

  17. 17

    Barter, P.J. et al. Antiinflammatory properties of HDL. Circ. Res. 95, 764–772 (2004).

  18. 18

    Hemmi, H. et al. A Toll-like receptor recognizes bacterial DNA. Nature 408, 740–745 (2000).

  19. 19

    Sparwasser, T. et al. Macrophages sense pathogens via DNA motifs: induction of tumor necrosis factor-alpha-mediated shock. Eur. J. Immunol. 27, 1671–1679 (1997).

  20. 20

    Ulevitch, R.J. & Johnston, A.R. The modification of biophysical and endotoxic properties of bacterial lipopolysaccharides by serum. J. Clin. Invest. 62, 1313–1324 (1978).

  21. 21

    Levine, D.M., Parker, T.S., Donnelly, T.M., Walsh, A. & Rubin, A.L. In vivo protection against endotoxin by plasma high density lipoprotein. Proc. Natl. Acad. Sci. USA 90, 12040–12044 (1993).

  22. 22

    Wurfel, M.M., Hailman, E. & Wright, S.D. Soluble CD14 acts as a shuttle in the neutralization of lipopolysaccharide (LPS) by LPS-binding protein and reconstituted high density lipoprotein. J. Exp. Med. 181, 1743–1754 (1995).

  23. 23

    Triantafilou, M., Miyake, K., Golenbock, D.T. & Triantafilou, K. Mediators of innate immune recognition of bacteria concentrate in lipid rafts and facilitate lipopolysaccharide-induced cell activation. J. Cell Sci. 115, 2603–2611 (2002).

  24. 24

    Zhu, X. et al. Macrophage ABCA1 reduces MyD88-dependent Toll-like receptor trafficking to lipid rafts by reduction of lipid raft cholesterol. J. Lipid Res. 51, 3196–3206 (2010).

  25. 25

    Fulton, D.L. et al. TFCat: the curated catalog of mouse and human transcription factors. Genome Biol. 10, R29 (2009).

  26. 26

    Horton, J.D. et al. Combined analysis of oligonucleotide microarray data from transgenic and knockout mice identifies direct SREBP target genes. Proc. Natl. Acad. Sci. USA 100, 12027–12032 (2003).

  27. 27

    Heinz, S. et al. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol. Cell 38, 576–589 (2010).

  28. 28

    Zimarino, M. et al. Optical coherence tomography accurately identifies intermediate atherosclerotic lesions—an in vivo evaluation in the rabbit carotid artery. Atherosclerosis 193, 94–101 (2007).

  29. 29

    Carmeliet, P. et al. Vascular wound healing and neointima formation induced by perivascular electric injury in mice. Am. J. Pathol. 150, 761–776 (1997).

  30. 30

    Zimmer, S. et al. Activation of endothelial toll-like receptor 3 impairs endothelial function. Circ. Res. 108, 1358–1366 (2011).

  31. 31

    Seetharam, D. et al. High-density lipoprotein promotes endothelial cell migration and reendothelialization via scavenger receptor-B type I. Circ. Res. 98, 63–72 (2006).

  32. 32

    Petoumenos, V., Nickenig, G. & Werner, N. High-density lipoprotein exerts vasculoprotection via endothelial progenitor cells. J. Cell Mol. Med. 13, 4623–4635 (2009).

  33. 33

    Speer, T. et al. Abnormal high-density lipoprotein induces endothelial dysfunction via activation of Toll-like receptor-2. Immunity 38, 754–768 (2013).

  34. 34

    Saemann, M.D. et al. The versatility of HDL: a crucial anti-inflammatory regulator. Eur. J. Clin. Invest. 40, 1131–1143 (2010).

  35. 35

    Michelsen, K.S. et al. Lack of Toll-like receptor 4 or myeloid differentiation factor 88 reduces atherosclerosis and alters plaque phenotype in mice deficient in apolipoprotein E. Proc. Natl. Acad. Sci. USA 101, 10679–10684 (2004).

  36. 36

    Mullick, A.E., Tobias, P.S. & Curtiss, L.K. Modulation of atherosclerosis in mice by Toll-like receptor 2. J. Clin. Invest. 115, 3149–3156 (2005).

  37. 37

    Kim, T.W. et al. The critical role of IL-1 receptor-associated kinase 4-mediated NF-kappaB activation in modified low-density lipoprotein-induced inflammatory gene expression and atherosclerosis. J. Immunol. 186, 2871–2880 (2011).

  38. 38

    Kleemann, R., Zadelaar, S. & Kooistra, T. Cytokines and atherosclerosis: a comprehensive review of studies in mice. Cardiovasc. Res. 79, 360–376 (2008).

  39. 39

    Gold, E.S. et al. ATF3 protects against atherosclerosis by suppressing 25-hydroxycholesterol-induced lipid body formation. J. Exp. Med. 209, 807–817 (2012).

  40. 40

    Yvan-Charvet, L. et al. ATP-binding cassette transporters and HDL suppress hematopoietic stem cell proliferation. Science 328, 1689–1693 (2010).

  41. 41

    Lerch, P.G., Spycher, M.O. & Doran, J.E. Reconstituted high density lipoprotein (rHDL) modulates platelet activity in vitro and ex vivo. Thromb. Haemost. 80, 316–320 (1998).

  42. 42

    Murphy, A.J. et al. High-density lipoprotein reduces the human monocyte inflammatory response. Arterioscler. Thromb. Vasc. Biol. 28, 2071–2077 (2008).

  43. 43

    Lerch, P.G., Fortsch, V., Hodler, G. & Bolli, R. Production and characterization of a reconstituted high density lipoprotein for therapeutic applications. Vox Sang. 71, 155–164 (1996).

  44. 44

    Havel, R.J., Eder, H.A. & Bragdon, J.H. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J. Clin. Invest. 34, 1345–1353 (1955).

  45. 45

    Thelen, K.M., Laaksonen, R., Paiva, H., Lehtimaki, T. & Lutjohann, D. High-dose statin treatment does not alter plasma marker for brain cholesterol metabolism in patients with moderately elevated plasma cholesterol levels. J. Clin. Pharmacol. 46, 812–816 (2006).

  46. 46

    Crameri, A. et al. The role of seladin-1/DHCR24 in cholesterol biosynthesis, APP processing and Abeta generation in vivo. EMBO J. 25, 432–443 (2006).

  47. 47

    Fulton, D.L. et al. TFCat: the curated catalog of mouse and human transcription factors. Genome Biol. 10, R29 (2009).

  48. 48

    Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl. Acad. Sci. USA 102, 15545–15550 (2005).

  49. 49

    Langmead, B., Trapnell, C., Pop, M. & Salzberg, S.L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).

  50. 50

    Heinz, S. et al. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol. Cell 38, 576–589 (2010).

  51. 51

    Zhang, Y. et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9, R137 (2008).

Download references

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.

Author information

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.

Correspondence to Eicke Latz.

Ethics declarations

Competing interests

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)

A summary of all microarray experiments performed for this study.

(XLSX 15 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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) doi:10.1038/ni.2784

Download citation

Further reading