Abstract

Innate immune memory is a vital mechanism of myeloid cell plasticity that occurs in response to environmental stimuli and alters subsequent immune responses. Two types of immunological imprinting can be distinguished—training and tolerance. These are epigenetically mediated and enhance or suppress subsequent inflammation, respectively. Whether immune memory occurs in tissue-resident macrophages in vivo and how it may affect pathology remains largely unknown. Here we demonstrate that peripherally applied inflammatory stimuli induce acute immune training and tolerance in the brain and lead to differential epigenetic reprogramming of brain-resident macrophages (microglia) that persists for at least six months. Strikingly, in a mouse model of Alzheimer’s pathology, immune training exacerbates cerebral β-amyloidosis and immune tolerance alleviates it; similarly, peripheral immune stimulation modifies pathological features after stroke. Our results identify immune memory in the brain as an important modifier of neuropathology.

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Acknowledgements

We thank P. Rizzu for experimental advice, L. Walker for manuscript comments and D. Bryce for statistical advice. This study was supported by a PhD fellowship from the Studienstiftung des Deutschen Volkes (A.-C.W.), a Roman Herzog Fellowship from the Hertie Foundation (J.J.N.), and grants from the network ‘Neuroinflammation in Neurodegeneration’ (State of Baden-Wuerttemberg, Germany; M.J. and M.P.), the Sobek-Stiftung (M.P.), the DFG (SFB992, Reinhart-Koselleck-Grant to M.P., SFB704 to J.L.S.), the European Research Council (A.F.) the Fortüne Program (Med. Faculty, Univ. Tuebingen; 2075-1-0; J.J.N.), the Fritz Thyssen Foundation (Cologne, Germany; J.J.N.) and the Paul G. Allen Family Foundation (Seattle, USA; J.J.N.). M.B. and J.L.S are members of the Excellence Cluster ImmunoSensation.

Author information

Author notes

  1. These authors contributed equally: Ann-Christin Wendeln, Karoline Degenhardt.

Affiliations

  1. German Center for Neurodegenerative Diseases (DZNE), Tübingen, Germany

    • Ann-Christin Wendeln
    • , Karoline Degenhardt
    • , Jessica Wagner
    • , Lisa M. Häsler
    • , Katleen Wild
    • , Angelos Skodras
    • , Matthias Staufenbiel
    • , Mathias Jucker
    •  & Jonas J. Neher
  2. Department of Cellular Neurology, Hertie Institute for Clinical Brain Research, University of Tübingen, Tübingen, Germany

    • Ann-Christin Wendeln
    • , Karoline Degenhardt
    • , Jessica Wagner
    • , Lisa M. Häsler
    • , Katleen Wild
    • , Angelos Skodras
    • , Matthias Staufenbiel
    • , Mathias Jucker
    •  & Jonas J. Neher
  3. Graduate School of Cellular and Molecular Neuroscience, University of Tübingen, Tübingen, Germany

    • Ann-Christin Wendeln
    • , Karoline Degenhardt
    •  & Jessica Wagner
  4. Department of Psychiatry and Psychotherapy, University Medical Center Göttingen, Göttingen, Germany

    • Lalit Kaurani
    • , Michael Gertig
    •  & André Fischer
  5. Department for Systems Medicine and Epigenetics in Neurodegenerative Diseases, German Center for Neurodegenerative Diseases (DZNE), Göttingen, Germany

    • Lalit Kaurani
    • , Michael Gertig
    • , Gaurav Jain
    • , Tonatiuh Pena Centeno
    • , Vincenzo Capece
    • , Md. Rezaul Islam
    • , Cemil Kerimoglu
    •  & André Fischer
  6. Genomics and Immunoregulation, LIMES-Institute, University of Bonn, Bonn, Germany

    • Thomas Ulas
    •  & Joachim L. Schultze
  7. Bioinformatics Unit, German Center for Neurodegenerative Diseases (DZNE), Göttingen, Germany

    • Gaurav Jain
    • , Tonatiuh Pena Centeno
    •  & Vincenzo Capece
  8. Institute of Neuropathology, Faculty of Medicine, University of Freiburg, Freiburg, Germany

    • Thomas Blank
    • , Ori Staszewski
    • , Moumita Datta
    •  & Marco Prinz
  9. Platform for Single Cell Genomics and Epigenomics at the University of Bonn and the German Center for Neurodegenerative Diseases, Bonn, Germany

    • Joachim L. Schultze
  10. Molecular Immunology in Neurodegeneration, German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany

    • Marc Beyer
  11. BIOSS Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany

    • Marco Prinz

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Contributions

K.D., A.-C.W., J.W., L.M.H., K.W., A.S., T.B, O.S., M.D. and J.J.N. performed microglial isolation, in vivo and ex vivo experiments and histological/biochemical analyses. M.G., L.K., G.J., T.P.C., V.C., R.I., C.K., A.F., M.B., T.U., J.L.S. and J.J.N. performed ChIP–seq and RNA-seq analyses. J.J.N conceived the study and coordinated the experiments together with M.J., A.F., M.P., M.B., J.L.S. and M.S. J.J.N. wrote the manuscript, with contributions from all authors.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Jonas J. Neher.

Extended data figures and tables

  1. Extended Data Fig. 1 Acute responses to LPS injections.

    a, Weight changes after injection of LPS (wild-type mice: n = 11, 11, 11, 11, 4 from left to right for PBS, n = 9, 9, 9, 8, 7 for 1 × LPS, n = 10, 10, 10, 10, 7 for 4 × LPS; APP animals: n = 14, 14, 14, 14, 7 for PBS, n = 8, 8, 8, 5, 5 for 1 × LPS; n = 10, 10, 10, 10, 10 for 4 × LPS; Cre mice: n = 5, 5, 4). b, c, Morphological changes in microglia (n = 6, 6, 6, 6, 6 mice). Scale bar, 50 µm. d, Numbers of microglia and activated (GFAP+) astrocytes (microglia: n = 6, 7, 8, 6, 6 mice, astrocytes: n = 6, 8, 9, 7, 5 mice). e, Blood and brain levels of LPS after daily injections with 500 µg per kg bodyweight (n = 4, 3, 3, 3, 3 animals). f, Assessment of iron entry from the blood (detected by Prussian blue staining) shows positive staining in an aged (>25 months) APP transgenic mouse, but not after repeated intraperitoneal LPS injections (n = 3 mice analysed). g, In heterozygous mice expressing red fluorescent protein (RFP) under the type 2 CC chemokine receptor (Ccr2) promoter, no entry of CCR2-expressing blood monocytes was detected after repeated LPS injection (staining for RFP; insert shows RFP-positive monocytes in the choroid plexus; n = 3 mice analysed). Scale bar, 100 µm. Data are means ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 for one-way ANOVA with Tukey correction. Source data

  2. Extended Data Fig. 2 Cytokine response after acute LPS injections.

    a, Additional cytokines (Fig. 1) analysed in the serum (top) and brain (bottom) 3 h after each daily intraperitoneal LPS injection on four consecutive days in 3-month-old mice (control mice received PBS injections; n = 16, 11, 12, 9, 9, 7, 7 and 5, 13, 4, 6, 9, 4, 5 mice for groups from left to right). b, c, Cytokine response in the blood only in wild-type (b, n = 6, 7, 8, 5, 5, 3, 3 mice) or APP23 mice (c, n = 10, 3, 3, 3, 4, 3, 3 mice). d, e, Cytokine response in the brain only in wild-type (d, n = 6, 7, 8, 5, 5, 3, 3 mice) or APP23 mice (e, n = 10, 4, 4, 4, 4, 4, 4 mice). Data are means ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 for independent-samples median test with correction for multiple comparisons. Source data

  3. Extended Data Fig. 3 APP levels and processing, neuritic dystrophy and astrocyte activation in 9-month-old APP23 animals.

    a, b, Micrograph of fluorescent staining for amyloid plaque (Methoxy-X04; green) and amyloid precursor protein (APP; red) (a) shows neuritic dystrophy surrounding the amyloid deposit, which is unchanged by LPS treatments (b; n = 5, 5, 5 animals). c, Overall Pearson’s correlation of plaque size with neuritic dystrophy (APP area; n = 49, 39, 42 plaques for PBS, 1 × LPS, 4 × LPS groups). d, Western blotting analysis (for gel source data, see Supplementary Fig. 1) of brain homogenates for APP and C-terminal fragment-β (CTFβ; n = 7, 4, 7 mice), and soluble APPβ ELISA (n = 6, 6, 6 mice). e, Micrograph of activated astrocytes (glial fibrillar acidic protein: GFAP) surrounding an amyloid plaque (Congo red) and quantification of the number of plaque-associated GFAP-positive astrocytes (n = 6, 6, 5 mice). Scale bar, 10 µm (a), 20 µm (e). Data are means ± s.e.m. *P < 0.05 for one-way ANOVA with Tukey correction. Source data

  4. Extended Data Fig. 4 Cytokine levels in 9-month-old animals.

    a, Cytokine measurements in brain homogenates of 9-month-old wild-type (n = 8, 8, 7 mice) and APP23 mice (n = 14, 10, 10 mice) treated i.p. with 1 × LPS or 4 × LPS at 3 months of age. b, Cytokine measurements in the serum of 9-month-old wild-type (WT; n = 14, 9, 13 mice) and APP23 mice (APP; n = 18, 12, 14 mice) after i.p. stimulation with 1 × LPS or 4 × LPS at 3 months of age. c, Cytokine measurements in the serum of wild-type mice stimulated i.p. with 1 × LPS or 4 × LPS at 3 months of age and re-stimulated with an additional LPS injection (500 µg kg–1) at 9 months of age (n = 10, 7, 10 animals). Data are means ± s.e.m. *P < 0.05, **P < 0.01 for two-way ANOVA with Tukey correction. In b a significant main effect for genotype is indicated by bars spanning all conditions of the same genotype. Source data

  5. Extended Data Fig. 5 Cytokine levels after brain ischaemia and in blood of 4-month-old mice.

    Three-month-old animals were injected i.p. with 1 × LPS or 4 × LPS and incubated for 4 weeks before receiving a stroke. a, Cytokine measurements in brain homogenates 24 h after stroke (n = 5, 7, 5, 5 animals). b, Cytokine measurements in the serum (n = 6, 6, 6 animals). Data are means ± s.e.m. ***P < 0.001 for one-way ANOVA with Tukey correction. Source data

  6. Extended Data Fig. 6 Microglial sorting strategy.

    Microglia were sorted as CD11bhigh and CD45low cells (population P4) from 9-month-old APP23 mice or wild-type littermates following i.p. injections of 1 × LPS or 4 × LPS at 3 months of age.

  7. Extended Data Fig. 7 Analysis of microglial enhancers.

    Microglial enhancers were analysed in 9-month-old wild-type and APP23 (APP) mice treated intraperitoneally with 1 × LPS or 4 × LPS at 3 months of age. a, Exemplary UCSC browser images of genomic region around the Hif1a gene (normalized to input and library dimension). b, Numbers of regions with differentially regulated H3K4me1 levels. c, Heatmaps of H3K4me1 regions (centred on H3K27ac peaks). d, Pairwise correlations between the two replicates of H3K4me1 read densities in differentially regulated regions. eg, Analyses of H3K27ac levels analogous to bd for H3K4me1. n = 2 replicates (8–10 mice per replicate); differential enhancers showed a cumulative Poisson P < 0.0001.

  8. Extended Data Fig. 8 Transcription factor motif analysis of active enhancer regions.

    Motif analysis was performed for selected conditions to identify transcription factors involved in the differential activation of enhancers (using putative enhancer regions present in both replicates within 500 bp around enhancer peaks). a, For all active enhancers, motif analysis was performed using the union H3K27ac peak file and standard background (random genomic sequence). b, Pairwise comparisons between conditions, using the first condition’s H3K27ac peak file as input and the second condition’s peak file as background. As motif enrichment was often relatively low, the analysis was focused on transcription factor (families), whose motifs occurred at least twice in ‘known’ (black) and ‘de novo’ motifs (blue). Motifs are identified by HOMER software using hypergeometric testing (no adjustment for multiple comparisons was made).

  9. Extended Data Fig. 9 Peripherally applied cytokines induce immune memory in the brain.

    a, Experimental design. b, Cytokine responses in the brain, four weeks after peripheral cytokine application (n = 17, 5, 5, 21, 8, 8, 15 mice from left to right). Note that TNF dose-dependently enhances (low dose) or decreases (high dose) certain cytokines. Similar to high dose TNF, certain cytokines are also reduced by peripheral application of IL-10 four weeks earlier. c, Cytokine responses in the periphery are unaffected (n = 8, 21, 9, 5,10 mice). Data are means ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 for one-way ANOVA with Tukey correction. Source data

Supplementary information

  1. Reporting Summary

  2. Supplementary Figure

    Gel source data for western blot in Extended Data Fig. 3

  3. Supplementary Table 1

    Differential microglial H3K4me1 levels

  4. Supplementary Table 2

    Differential microglial H3K27ac levels

  5. Supplementary Table 3

    Microglial gene expression matrix

  6. Supplementary Table 4

    WGCNA analysis

  7. Video 1: Effect of microglia-specific Tak1 knockout on sickness behaviour

    Microglia-specific (CX3CR1-CreER induced) knockout of transforming growth factor-β-activated kinase 1 (Tak1) alleviates sickness behaviour in mice 3 h after injection of the second dose of LPS. CX3CR1-Cre+ mice at the end of the movie are the first and third mice from the left

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https://doi.org/10.1038/s41586-018-0023-4

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