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A new type of microglia gene targeting shows TAK1 to be pivotal in CNS autoimmune inflammation

Nature Neuroscience volume 16, pages 1618–1626 (2013)Cite this article

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Abstract

Microglia are brain macrophages and, as such, key immune-competent cells that can respond to environmental changes. Understanding the mechanisms of microglia-specific responses during pathologies is hence vital for reducing disease burden. The definition of microglial functions has so far been hampered by the lack of genetic in vivo approaches that allow discrimination of microglia from closely related peripheral macrophage populations in the body. Here we introduce a mouse experimental system that specifically targets microglia to examine the role of a mitogen-associated protein kinase kinase kinase (MAP3K), transforming growth factor (TGF)-β-activated kinase 1 (TAK1), during autoimmune inflammation. Conditional depletion of TAK1 in microglia only, not in neuroectodermal cells, suppressed disease, significantly reduced CNS inflammation and diminished axonal and myelin damage by cell-autonomous inhibition of the NF-κB, JNK and ERK1/2 pathways. Thus, we found TAK1 to be pivotal in CNS autoimmunity, and we present a tool for future investigations of microglial function in the CNS.

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Figure 1: Characterization of the Cx3cr1Cre line in the CNS.
Figure 2: Long-term microglial specificity in Cx3cr1CreER mice.
Figure 3: Microglia-restricted absence of TAK1 abolishes autoimmune inflammation in the CNS.
Figure 4: Reduced CNS damage and immune suppression in Cx3cr1CreER:Tak1fl/fl mice.
Figure 5: Diminished MHC class II expression in Cx3cr1CreER:Tak1fl/fl mice during EAE.
Figure 6: Microglia-specific TAK1 controls cell-intrinsic activation of NF-κBp65.

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Acknowledgements

We thank M. Oberle, C. Fix and S. Gaupp for excellent technical assistance. Special thanks to M. Olschewski for help with the thorough statistical analysis of our data. M.P. was supported by the Bundesministerium für Bildung und Forschung–funded competence network of multiple sclerosis (KKNMS), the competence network of neurodegenerative disorders (KNDD), the Deutsche Forschungsgemeinschaft (SFB 992, FOR1336, PR 577/8-1), the Fritz-Thyssen Foundation, the Gemeinnützige Hertie Foundation (GHST) and Biogen Idec. P.F.M. was supported by an MD educational grant of the SFB620. M.H. was funded by the Helmholtz Foundation, the SFB-TR36, a European Research Council starting grant and the Helmholtz Alliance Preclinical Comprehensive Cancer Center. S.J. was supported by the Deutsche Forschungsgemeinschaft (FOR1336) and by the Israel Science Foundation.

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Author notes

  1. Tobias Goldmann, Peter Wieghofer, Steffen Jung and Marco Prinz: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute of Neuropathology, University of Freiburg, Freiburg, Germany

    Tobias Goldmann, Peter Wieghofer, Philippe F Müller, Stefanie M Brendecke, Katrin Kierdorf, Ori Staszewski, Moumita Datta & Marco Prinz

  2. Faculty of Biology, University of Freiburg, Freiburg, Germany

    Peter Wieghofer

  3. Department of Immunology, The Weizmann Institute of Science, Rehovot, Israel

    Yochai Wolf, Diana Varol, Simon Yona & Steffen Jung

  4. Department of Internal Medicine III, University Hospital Rheinisch-Westfälische Technische Hochschule,

    Tom Luedde

  5. Aachen, Aachen, Germany

    Tom Luedde

  6. Institute of Virology, Technische Universität München/Helmholtz Zentrum München, Munich, Germany

    Mathias Heikenwalder

  7. BIOSS Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany

    Marco Prinz

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Contributions

T.G., P.W., P.F.M., S.M.B., K.K., D.V., Y.W., O.S. and M.D. conducted experiments. S.Y. generated the transgenic mice. S.J., M.H. and T.L. contributed to the in vivo studies and provided mice or reagents. M.P. and S.J. supervised the project and wrote the manuscript.

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Correspondence to Steffen Jung or Marco Prinz.

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Integrated supplementary information

Supplementary Figure 1 Microglial rearrangement of individual conditional yfp and rfp reporter alleles in Cx3cr1CreER:R26-yfp:R26-rfp mice under different TAM administration routes.

(a) Breeding scheme of CX3CR1CreER animals with R26-yfp:R26-rfp indicator mice. (b) Flow cytometry analysis of the blood for YFP and RFP. For s.c. injections, mice received dosages of 10 mg on 4 consecutive days and were analyzed at day 14. For oral administration by gavage, mice received doses of 5 mg on 6 consecutive days and were analyzed at day 14. Results are representative of three independent experiments.

Supplementary Figure 2 Long term and stable microglia-specific gene activation in Cx3cr1CreER:R26-yfp mice.

Induction but subsequent progressive loss of cells harbouring gene rearrangements in peripheral myeloid cells (Ly6Clo monocytes, splenic CD4+ DCs) and tissue macrophages (Kupffer cells) but persistence of genomic modification in microglia. Representative flow cytometry data displaying different recombination efficacies in distinct myeloid cell types. Results are representative of two independent experiments.

Supplementary Figure 3 Microglia can be distinguished from infiltrating myeloid cells in Cx3cr1CreER:R26-yfp mice during autoimmune inflammation.

(a) Scheme of EAE induction in CX3CR1CreER:R26-yfp mice. (b) Spinal cord flow cytometric analysis reveals the presence of YFP+ microglia that can be separated from non-labelled myeloid cells from the blood during MOG35-55-induced CNS inflammation. One representative experiment out of two is shown. (c) Quantification thereof. Mean ± SEM per group are depicted. Results are representative of two independent experiments.

Supplementary Figure 4 Low gene recombination efficacy in microglia of LysMCre animals.

(a) Flow cytometry analysis of percoll gradient-isolated microglia from LysMCre:R26-yfp mice (left, Cre negative litter is shown as dotted line) and quantification thereof (right). Mean ± SEM per group are depicted. (B) Direct fluorescence microscopic visualization revealed few YFP-positive ramified cells (green) with typical microglial morphology and Iba-1 immunoreactivity (red) in some regions of the brain whereas most microglia were not YFP-positive (asterisks). Arrows point to double positive cells. Scale bar = 20 μm. (c) Semi-quantitative evaluation of YFP-Iba-1 double positive microglia in distinct regions of the CNS. Data are represented as mean ± SEM of three to four mice per group. (d) YFP-positive cells in different blood cell subsets. Data are represented as mean ± SEM of four to six mice per group.

Supplementary Figure 5 Limited target gene activation in microglia of CD11cCre animals.

(a) FACS analysis on isolated CD45loCD11b+ microglia in CD11cCre:R26-yfp mice (left, a Cre negative litter is depicted as dotted line) and quantification thereof (right). Mean ± SEM per group are shown. (b) Iba-1 (red) and YFP immunohistochemistry (green) in different regions of the brain in CD11cCre:R26-yfp mice. Asterisks indicate the localization of Iba1-positive cells. Scale bar = 20 μm. (c) Semi-quantitative evaluation of YFP-Iba-1 double positive microglia on histological slices in distinct regions of the CNS. Data are represented as mean ± SEM of at least three mice per group. (d) Percentage of YFP-positive cells in different blood cell subsets. Data are represented as mean ± SEM of five mice per group.

Supplementary Figure 6 Microglia-specific TAK1 is dispensable for normal CNS homeostasis.

Absence of any gross abnormalities within the brain (a) and spinal cord (b) in the absence of TAK1 in microglia. H&E staining revealed unaltered structures in the cortex and spinal cord in CX3CR1CreER:Tak1fl/fl mice four weeks after TAM injection into five to seven week old animals. Scale bars = 500 μm (overview) and 100 μm (detail). Middle panel: Iba-1 immunohistochemistry revealed no microglia clusters or malformed microglia (scale bars = 25 μm (overview) and 20 μm (detail)). Lower panel: GFAP immunohistochemistry exhibited no signs of astrogliosis in CX3CR1CreER:Tak1fl/fl animals.

Supplementary Figure 7 No signs of cell activation in microglia devoid of TAK1.

Microglia (Iba-1, green) in CX3CR1CreER:Tak1fl/fl animals do not express MHC class II or Lamp2 (red, scale bar = 25 μm). Inserts show double positive perivascular macrophages in the same sections as control.

Supplementary Figure 8 Impaired ERK, p38 and JNK activation in microglia in the CNS of Cx3cr1CreER:Tak1fl/fl mice with EAE 20 d post-immunization.

Frozen sections of lumbal spinal cords from microglia-specific CX3CR1CreER:Tak1fl/fl mice with EAE, stained for IB4 (red) for microglia with distinct processes and activated pERK1/2, pp38 or pJNK (green), respectively. Nuclei are stained with DAPI (4,6-diamidino-2-phenylindole; blue). Scale bar = 10 μm. Arrows highlight double positive microglia. Asterisks indicate an activated hematopoietic cell. Results are representative of two independent experiments.

Supplementary Figure 9 High gene recombination efficacy in primary microglia of Cx3cr1CreER mice.

(a) Scheme for the induction of recombination in primary microglia from CX3CR1CreER:R26-yfp animals. Hydroxy-tamoxifen (OH-TAM) was applied three days prior to analysis. (b) Primary microglia in CX3CR1CreER:R26-yfp mice were characterized by surface expression of CD45 and CD11b and concomitant YFP expression three days after OH-TAM application by FACS. The percentage of YFP+ microglia is indicated. A Cre negative littermate is shown as a grey line. (c) High gene recombination in primary microglia from CX3CR1CreER:R26-yfp individuals using immunohistochemistry for YFP (green), the microglia marker Iba-1 (red), DAPI (blue) after OH-TAM challenge. Virtually all microglia from CX3CR1CreER:R26-yfp mice were EYFP+ whereas no microglia were YFP+ in R26-yfp mice.

Supplementary Figure 10 Full-length western blot images of Figures 3a,c and 6d.

Supplementary Figure 11 Full-length images of Figure 6e.

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Goldmann, T., Wieghofer, P., Müller, P. et al. A new type of microglia gene targeting shows TAK1 to be pivotal in CNS autoimmune inflammation. Nat Neurosci 16, 1618–1626 (2013). https://doi.org/10.1038/nn.3531

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