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ApoE attenuates unresolvable inflammation by complex formation with activated C1q

Nature Medicine volume 25, pages 496–506 (2019)Cite this article

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Abstract

Apolipoprotein-E (ApoE) has been implicated in Alzheimer’s disease, atherosclerosis, and other unresolvable inflammatory conditions but a common mechanism of action remains elusive. We found in ApoE-deficient mice that oxidized lipids activated the classical complement cascade (CCC), resulting in leukocyte infiltration of the choroid plexus (ChP). All human ApoE isoforms attenuated CCC activity via high-affinity binding to the activated CCC-initiating C1q protein (KD~140–580 pM) in vitro, and C1q–ApoE complexes emerged as markers for ongoing complement activity of diseased ChPs, Aβ plaques, and atherosclerosis in vivo. C1q–ApoE complexes in human ChPs, Aβ plaques, and arteries correlated with cognitive decline and atherosclerosis, respectively. Treatment with small interfering RNA (siRNA) against C5, which is formed by all complement pathways, attenuated murine ChP inflammation, Aβ-associated microglia accumulation, and atherosclerosis. Thus, ApoE is a direct checkpoint inhibitor of unresolvable inflammation, and reducing C5 attenuates disease burden.

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Fig. 1: ChP lipid, inflammation, and interferon signatures.
Fig. 2: Complement affects ChP inflammation.
Fig. 3: ApoE inhibits CCC initiation by high-affinity binding to C1q.
Fig. 4: ApoE or ApoE139–152 binds to the C1q stalk.
Fig. 5: ChP C1q–ApoE complexes correlate with cognitive decline in Alzheimer’s disease.
Fig. 6: C1q–ApoE complexes are indicative of CCC activity in atherosclerosis.

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Data availability

All microarray data can be found under GEO database accession number GSE85774 and GSE85775 for ChPs. Microarray data for aorta had been published previously with the accession number GSE40156 (ref. 57). The remaining source data for figures in the manuscript will be made available upon reasonable request.

Change history

  • 04 February 2019

    In the version of this article originally published, a sentence was erroneously included in the author contributions, and information regarding second shared authorship was missing from the author contributions. The following should not have been included in the author contributions: “C.W. and A.J.R.H. supervised the work presented in Figs. 1, 2, 5, 6; P.Z. and C.S. supervised the work presented in Figs. 3, 4.” Additionally, this sentence should have appeared at the beginning of the author contributions: “These authors contributed equally: C.W., P.F.Z., C.S., and A.J.R.H.” The errors have been corrected in the print, PDF and HTML versions of the article.

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Acknowledgements

We thank W. Schneider, Medical University of Vienna, Austria, for advice; T. Hallström, Y. Lin, and S. Hälbich, Leibniz Institute for Natural Product Research and Infection Biology, Jena, for performing complement assays; W. Wilfert, Institute of Laboratory Medicine, University of Munich for lipid analyses; S. Schmidt, Institute for Experimental Neurology, Jena, for advice; and N. Buresch, Center for Neuropathology and Prion Research, Munich, for technical assistance; M. Jucker, Hertie Institute for Clinical Brain Research, University of Tübingen, for experimental support. This work was funded by the Deutsche Forschungsgemeinschaft (DFG): YI 133/2-1 to C.Y.; HA 1083/15-4 to A.J.R.H.; MO 3054/1-1 to S.M.; The German Collaborative Research Center (CRC124/2-C4), SK46/2 to C.S.; CRC124/2-C6 and DFG TR 1992 to P.F.Z.; the German Centre for Cardiovascular Research (DZHK MHA VD1.2), DFG SFB 1123/A1 and Z3, the European Research Council (ERC AdG 692511) to C.W.; SFB1123/Z01, INST409/150-1 FUGG to R.T.A.M.; Chinese National Natural Science Foundation (31770983) to D.H.; S.A. and L.D.H. are doctoral researchers at the International Leibniz Research School (ILRS) in Jena.

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Author notes

  1. These authors contributed equally: Changjun Yin, Susanne Ackermann.

Authors and Affiliations

  1. Institute for Cardiovascular Prevention (IPEK), Ludwig-Maximilians-University, Munich, Germany

    Changjun Yin, Zhe Ma, Sarajo K. Mohanta, Chuankai Zhang, Yuanfang Li, Remco T. A. Megens, Sabine Steffens, Christian Weber & Andreas J. R. Habenicht

  2. German Center for Cardiovascular Research (DZHK), partner site Munich Heart Alliance, Munich, Germany

    Changjun Yin, Sabine Steffens & Christian Weber

  3. Leibniz Institute for Natural Product Research and Infection Biology, Jena, Germany

    Susanne Ackermann, Luke D. Halder, Peter F. Zipfel & Christine Skerka

  4. Centre for Electron Microscopy, Jena University Hospital, Friedrich-Schiller-University of Jena, Jena, Germany

    Sandor Nietzsche & Martin Westermann

  5. Department of Cardiovascular Medicine of Second Affiliated Hospital, Guizhou University of Traditional Chinese Medicine, Guiyang, China

    Li Peng

  6. Department of Integrated Traditional Chinese and Western Medicine, Union Hospital, Huazhong University of Science and Technology, Wuhan, China

    Desheng Hu

  7. Department of Surgery, University of Tennessee, Memphis, TN, USA

    Sai Vineela Bontha

  8. Cardiovascular Research Center (CVRC), University of Virginia, Charlottesville, VA, USA

    Prasad Srikakulapu

  9. Department of Information Technology, University Clinic Jena, Jena, Germany

    Michael Beer

  10. Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands

    Remco T. A. Megens & Christian Weber

  11. Institute for Anatomy II, University Clinic Jena, Jena, Germany

    Markus Hildner

  12. Department for Vascular and Endovascular Surgery, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany

    Hans-Henning Eckstein & Jaroslav Pelisek

  13. Center for Neuropathology and Prion Research, Ludwig-Maximilians-University, Munich, Germany

    Jochen Herms, Sigrun Roeber & Thomas Arzberger

  14. Munich Cluster of Systems Neurology (SyNergy), Ludwig-Maximilians-University, Munich, Germany

    Jochen Herms

  15. Department of Psychiatry and Psychotherapy, Ludwig-Maximilians-University, Munich, Germany

    Thomas Arzberger

  16. Alnylam Pharmaceuticals Cambridge, Cambridge, MA, USA

    Anna Borodovsky

  17. II. Medizinische Klinik und Poliklinik, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany

    Livia Habenicht

  18. Department of Laboratory Medicine, Medical University of Vienna and Center for Molecular Medicine (CeMM) of the Austrian Academy of Sciences, Vienna, Austria

    Christoph J. Binder

  19. Friedrich-Schiller-University, Faculty of Biological Sciences, Jena, Germany

    Peter F. Zipfel

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  1. Changjun Yin
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Contributions

These authors contributed equally: C.W., P.F.Z., C.S., and A.J.R.H.; C.Y. and A.J.R.H. designed and performed experiments and wrote the manuscript; S.A., P.F.Z., C.S. designed and performed experiments and contributed to writing the manuscript; C.W. provided critical intellectual input for experimental design and wrote the manuscript; A.B. provided the C5 siRNA; Z.M., S.K.M., C.Z., Y.L., S.N., M.W., L.P., D.H., S.V.B., P.S., M.B., R.T.A.M., S.S., M.H., L.D.H., H.-H.E., J.P., J.H., S.R., T.A., L.H., and C.J.B. performed experiments or analyzed the data.

Corresponding authors

Correspondence to Changjun Yin or Christine Skerka.

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Competing interests

C.Y., A.J.R.H., A.B., S.K.M., S.A., P.F.Z., and C.S. declare competing financial interests. C.Y., S.K.M., and A.J.R.H. are owners of Easemedcontrol R & D GmbH & Co KG Munich, Germany; A.B. is employed by Alnylam Pharmaceuticals Cambridge; Cambridge, MA, USA; C.Y. and A.J.R.H. have been named inventors on a pending patent application related to treatment and diagnosis of unresolvable inflammatory diseases (EP18183584.4); A.B. has been named as an inventor on patent applications related to C5 including PCT publication WO2014160129, and applications and patents based thereon; S.A., P.F.Z., and C.S. have been named inventors on a pending patent application (DE 10 2018 100 377.3).

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Extended data

Extended Data Fig. 1 Lipid deposits, BBB, and ChP gene signatures.

a, Vacuole (Va) represents lipid. Intercellular lipid (green) between two epithelial cells was quantified. 68 intercellular spaces from 3 ApoE−/− and 67 intercellular spaces from 3 WT mice were analyzed. Bar represents 1 µm. b, Lipid in ApoE−/− ChPs by TEM. Lymphocytes (left panel); macrophages/dendritic cells (DC) (middle panel); and ependymal cells contain lipid (right panel). Vacuole (Va) represents lipid. Bar 1 µm. c, ChPs were stained for cytokeratin (keratin, red) and leukocytes (CD45, green) (left panel); collagen IV (Co-IV, green) and CD68 (red) (middle panel). TEM shows single macrophage-foam cell/DCs adjacent to microvilli. Bar 10 µm. d, ChPs were stained with Ig (red) as described in Methods. Bars 10 µm. e, PFA-perfused brains were stained for Ig (Ig, red) and blood vessels (Col-IV, green) in the cerebellum. Perivascular Ig adjacent to blood vessels was quantified as described in Methods. WT (n = 3 mice); ApoE−/− (n = 3); ND ApoE3 (n = 3); HFD ApoE3 (n = 3); ND ApoE4 (n = 3); HFD ApoE4 (n = 3). Bar 10 µm. f, Laser capture microdissection (LCM)-based expression microarrays of ChPs. Heatmaps show transcript levels in GO terms immune system process, transcription factor binding, cell junction, and ATP binding. g, Genes that were down-regulated in ApoE−/− CPs and rescued either in ApoE3-KI and in ND or HFD ApoE4-KI mice. WT (n = 5 mice); ApoE−/− (n = 4); ND ApoE3 (n = 6); HFD ApoE3 (n = 6); ND ApoE4 (n = 6); HFD ApoE4 (n = 6). Data in c,d are representative images from at least 3 biologically independent mouse samples. Data in a,e,g represent means ± s.e.m. Two-tailed Student´s t test was applied to a,e,g. Gene names and statistics in supplementary Tables 1, 3.

Extended Data Fig. 2 Complement constituents in mouse ChPs.

a, ChPs were stained for C1q (red) and C4 (green). Bar 100 μm. b, C5 siRNA treatment blocks C5 protein deposition in ApoE−/− ChPs. c, ChPs were stained for C3. Ig represents lipid. d, Serum C3 and C5. Serum C3 and C5 protein levels were measured by ELISA. ApoE−/−(n = 6 mice), HFD ApoE4 (n = 5). e, High resolution confocal microscopy shows colocalization of ApoE4 (ApoE, red) and Ig (green, represents lipid) in HFD ApoE4-KI ChPs. ApoE−/− ChPs serve as negative controls for ApoE staining. f, Complement regulators are expressed in ChPs. WT (n = 5 mice); ApoE−/−(n = 4); ND ApoE3 (n = 6); HFD ApoE3 (n = 6); ND ApoE4 (n = 6); HFD ApoE4 (n = 6). g, ChP Factor H expressed between WT and ApoE−/− mice. WT (n = 5); ApoE−/−(n = 4.) h, ChP factor H protein in ChPs. White arrows indicate lipid positive areas. Data in a,b,c,e,h are representative images from at least 3 biologically independent mouse samples. Data in d,f,g represent means ± s.e.m. Two-tailed Student´s t test was applied to d,g; one-way ANOVA with Tukey posttest was applied to f; Gene names in supplementary Tabl. 3.

Extended Data Fig. 3 ApoE does not inhibit cleavage of C2 or C4 by C1s.

a, C1q binds immobilized malondialdehyde-modified LDL (MDA-LDL) and oxLDL but not native LDL or gelatin. b, ApoE isoforms in normal human serum (NHS) were added to MDA-LDL-coated microtiter plates and C4b deposition was determined by specific antisera. c,d IgM, MDA-LDL, and Aβ fibrils but not soluble Aβ activate complement and cause C3b deposition. BSA, gelatin as negative controls; e,f ApoE3 was incubated with either (e) C2 or (f) C4 in the presence of C1s. C2 and C4 were cleaved to their active forms C2a (α´30) and C4b (α´83) via C1s as revealed by the cleavage products in western blot analyses. g, ApoE3 has no cofactor activity for factor I in the cleavage of C4b to inactive iC4b. ApoE3 was incubated together with factor I, C4BP and C4b, and cleavage products were detected by western blot analysis as indicated (α´25 and α´13). Full scanned blot images in e,f,g are available from source data figures. Data in a-d represent means ± s.e.m. of three independent experiments. Two-tailed Student’s t test. Data in e,f,g are representative from 3 independent experiments.

Source data

Extended Data Fig. 4 ApoE binds to C1q but not to other complement components.

a, ApoE isoforms bind to the C1 complex, but not to C4 or C2. Biotinylated ApoE was immobilized on streptavidin-coated sensors and incubated with C1 complex, C4, C2, or buffer. b, The C1 complex binds to immobilized ApoE isoforms. c, ApoE isoforms bind to C1 and factor H, but not to C3 or C3b. d, NHS-derived C1 binds to immobilized plasma-purified ApoE3 and to recombinant ApoE isoforms. e, C1q binds to immobilized plasma-purified ApoE3 and to all recombinant ApoE isoforms. f, Plasma-purified C1q was coated on a sensor chip (CM5) and plasma-derived ApoE (62-1000 nM) was injected into the fluid phase (75 mM NaCl, 5 mM HEPES, 1 mM CaCl2). g, Mannose-binding lectin (MBL) does not bind to C1q as determined by biolayer interferometry. h, Apolipoprotein A (ApoA) does not bind to C1q as determined by biolayer interferometry. i, C1q-ApoE complexes revealed by proximity ligation assay (PLA) on cultured human apoptotic cells (THP-1) were detectable when treated with NHS but not with C1q-depleted serum (dNHS). Data represent mean fluorescence intensity (MFI) ± s.e.m. of 16 cells for each group. Bar 10 μm. Data in b,c,d,e represent means ± s.e.m. of at least three independent experiments. Data a,f,g and h represent means of at least two independent experiments. Two-tailed Student’s t test.

Extended Data Fig. 5 ApoE binds to the activated C1q; LDLR and C1sC1r tetramers do not compete with C1q-ApoE binding.

a, ApoE-C1q interaction is dependent on Ca2+. Real-time binding of ApoE to C1q was followed using biosensor analyses. Binding of ApoE to C1q is reduced in a dose-dependent manner upon increasing amounts of EGTA (0.1–3 mM). b,c, co-immunoprecipitation of C1q-ApoE complexes; (b) anti C1q antiserum precipitate C1q-ApoE complexes composed of purified proteins with activated C1q, but not with inactive C1q from NHS; (c) Anti-ApoE antiserum precipitates C1q-ApoE complexes but no complexes from NHS. C1q-ApoE complexes were eluted with glycine buffer, then, C1q or ApoE proteins were separated by SDS-PAGE and immunoblotted using goat anti-C1q antiserum (left panel of b, and c) or goat anti ApoE antiserum (right panel of b) separately. Full scanned blot images in b,c are available from source data figures. d, ApoE peptide P139-152 but not ApoE peptide P30-40 competes with immobilized ApoE3 for binding to C1q in a dose-dependent manner. e, C1q antibody binding to C1q is not affected by SDS. f, C1q and LDLR bind simultaneously to ApoE. 20 nM C1q was incubated with increasing concentrations of LDLR to immobilized ApoE and binding of C1q and LDLR was followed by ELISA. Background binding of anti C1q and anti LDLR antisera to immobilized ApoE were set as 0%. g, ApoE does not compete with C1sC1r tetramers for binding to C1q. C1q in addition to increasing amounts of C1sC1r tetramers was added to immobilized ApoE3 and C1q binding was determined. Data in d-g represent means ± s.e.m. of at least three independent experiments. Two-tailed Student´s t test. Data in a,b,c are representatives of 3 independent experiments.

Source data

Extended Data Fig. 6 Complement constituents in human ChPs and AD plaques.

a-b, Human ChP sections were stained for C1q (green) / C3 (red) (a) and C1q (green)/ApoE (red) (b). c,d, ChP sections were stained for CD68+ macrophages/DCs (c) and collagen IV (Col-IV) to mark basement membranes. Phase contrast shows lipid deposits in ChPs. e, ChP sections were stained for ApoE (green) and factor H (red); no primary antibody as control (NA). f,g, human brain sections were stained for Aβ (green) / ApoE (red) (left panels), Tau phosphorylation (pTau, green) / ApoE (red) (middle panels), and C1q (green) / ApoE (red) (right panels) (f). Blue for nuclei. No primary antibody as control (g). h, AD brain parenchyma sections were stained for C3 (red) / ApoE (green). Bar 100 μm for a-h; Data in a-h are representative images from at least 3 biologically independent samples.

Extended Data Fig. 7 Complement constituents in mouse brain.

a, 16-week old APPPS1-21 mouse brain sections were stained with Aβ/ApoE complexes (red) by PLA, methoxy X04 for Aβ plaque (blue). High resolution confocal images show the spatial location of Aβ-ApoE complexes and Aβ plaque in 3D view (lower panel). Bars represent 10 µm. b, Brain sections were stained with methoxy-X04, ApoE, and LAMP1; the size of areas covered by methoxy-X04, ApoE, and LAMP1 was determined. ApoE/X04 and LAMP1/X04 (X04 > 150 µm2) were quantified. n = 123 plaques from 4 control mice, 147 plaques from 5 treated mice. Bars 100 µm. c, Aβ plaque was stained with methoxy X04 (X04). Number of plaques per section and number of plaque per area were quantified. control (n = 4 mice), C5 (n = 5). Bar 1000 µm. d, Total plaque volume was determined in 3D, plaques were further grouped according to the plaque volume. n = 71 random fields from 4 control mice, 88 fields from 5 C5 siRNA treated mice. Bar 100 µm. e, 8-week old C57BL6 brain cortex sections were examined for the presence of C1q-ApoE complexes with methoxy X04. ApoE, or C1q only antisera were used as negative controls. Bar represents 10 µm. Data in a,e are representative images from at least 3 biologically independent mouse samples. Data in b,c,d represent means ± s.e.m. Two-tailed Student´s t test was applied to b,c,d; Two-way ANOVA was applied to c,d.

Extended Data Fig. 8 Complement and atherosclerosis.

a, Expression microarray analyses of aortas. Heatmaps show GO terms leukocyte migration, complement activation, phagocytosis, and cellular response to lipid. 6 weeks WT (n = 3 mice); 32 weeks WT (n = 3); 6 weeks ApoE−/− (n = 3); 32 weeks ApoE−/− (n = 3). b, aorta alternative complement pathway genes (factor B, factor H, factor D) mRNA expression in 6 weeks and 32 weeks old WT and ApoE−/− mouse aortas. 6 weeks WT (n = 3 mice); 32 weeks WT (n = 3); 6 weeks ApoE−/− (n = 3); 32 weeks ApoE−/− (n = 3). c,d, plasma cholesterol and body weight. e,f blood leukocytes and percentage. For c-f, control (n =11 mice); C5 siRNA (n =12 mice). g, blood CD4+ T cells, CD8+ T cells, and B220+ B cells by flow cytometry. Control (n = 6 mice); C5 siRNA (n = 6 mice). h, super-resolution microscopy shows colocalization of C1q (green) and ApoE (red) in human atherosclerotic plaque. Representative images from at least 3 biologically independent mouse samples. Bar 5 μm. Data in b,c,d,e,f,g represent means ± s.e.m. Two-tailed Student´s t test was applied to c.d.e.f.g; one-way ANOVA with Tukey posttest was applied to b; abbreviations: WBC, white blood cells; RBC, red blood cells; PLT, platelets; LYM, lymphocytes; MO, monocytes; GRA, granulocytes. Gene names and statistics in supplementary Table 7.

Extended Data Fig. 9 Graphical presentation of the body of in vivo data.

a, Pleiotropic impacts of single ApoE or single C1q molecules in brain as reported by others. Microglia cells are the major source of brain C1q. In response to Aβ plaques, resting microglia cells differentiate into plaque-associated microglia cells. Single actions of ApoE and C1q have been reported to be involved in multiple pathways as indicated in the Figure. Inactive C1q (yellow), activated C1q (red). b, Graphical presentation of the body of in vivo data. Three types of unresolvable inflammatory conditions were studied in 7 mouse models and in translational studies of human tissues, that is choroid plexus, aorta, and brain parenchyma.

Supplementary information

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Supplementary Tables

Supplementary Tables 1–8

Supplementary Video 1

Aβ plaque and C1q-ApoE complex in 3D.

Supplementary Video 2

Aβ plaque and Aβ-ApoE complex in 3D.

Source data

Source data Extended Data Fig. 3

Unprocessed Western Blots gels for extended figure 3.

Source data Extended Data Fig. 5

Unprocessed Western Blots gels for extended figure 5.

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Yin, C., Ackermann, S., Ma, Z. et al. ApoE attenuates unresolvable inflammation by complex formation with activated C1q. Nat Med 25, 496–506 (2019). https://doi.org/10.1038/s41591-018-0336-8

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