Abstract

Microglia and astrocytes modulate inflammation and neurodegeneration in the central nervous system (CNS)1,2,3. Microglia modulate pro-inflammatory and neurotoxic activities in astrocytes, but the mechanisms involved are not completely understood4,5. Here we report that TGFα and VEGF-B produced by microglia regulate the pathogenic activities of astrocytes in the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis. Microglia-derived TGFα acts via the ErbB1 receptor in astrocytes to limit their pathogenic activities and EAE development. Conversely, microglial VEGF-B triggers FLT-1 signalling in astrocytes and worsens EAE. VEGF-B and TGFα also participate in the microglial control of human astrocytes. Furthermore, expression of TGFα and VEGF-B in CD14+ cells correlates with the multiple sclerosis lesion stage. Finally, metabolites of dietary tryptophan produced by the commensal flora control microglial activation and TGFα and VEGF-B production, modulating the transcriptional program of astrocytes and CNS inflammation through a mechanism mediated by the aryl hydrocarbon receptor. In summary, we identified positive and negative regulators that mediate the microglial control of astrocytes. Moreover, these findings define a pathway through which microbial metabolites limit pathogenic activities of microglia and astrocytes, and suppress CNS inflammation. This pathway may guide new therapies for multiple sclerosis and other neurological disorders.

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Acknowledgements

This work was supported by grants NS087867, ES02530, AI126880 and AI093903 from the National Institutes of Health, RSG-14-198-01-LIB from the American Cancer Society and RG4111A1 and JF2161-A-5 from the National Multiple Sclerosis Society to F.J.Q. F.J.Q. and J.A. received support from International Progressive Multiple Sclerosis Alliance grant PA-1604-08459. V.R. received support from an educational grant from Mallinkrodt Pharmaceuticals (A219074) and by a fellowship from the German Research Foundation (DFG RO4866 1/1). M.P. is supported by the BMBF-funded competence network of multiple sclerosis (KKNMS), the Sobek-Stiftung and the DFG (SFB 992, SFB1140, SFB/TRR167, Reinhart-Koselleck-Grant) and the Ministry of Science, Research and the Arts, Baden-Wuerttemberg (Sonderlinie ‘Neuroinflammation’). Human fetal tissue came from the Human Fetal Tissue Repository (Albert Einstein College of Medicine) and from the University of Washington Birth Defects Research Laboratory (BDRL, Project Number 5R24HD000836-51).

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Nature thanks S. Liddelow, M. Platten, H. Wekerle and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Affiliations

  1. Ann Romney Center for Neurologic Diseases, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA

    • Veit Rothhammer
    • , Davis M. Borucki
    • , Emily C. Tjon
    • , Maisa C. Takenaka
    • , Chun-Cheih Chao
    • , Kalil Alves de Lima
    • , Cristina Gutiérrez-Vázquez
    • , Patrick Hewson
    • , Tradite Neziraj
    • , Matilde Borio
    • , Michael Wheeler
    •  & Francisco J. Quintana
  2. Institute of Neuropathology, Medical Faculty, University of Freiburg, Freiburg, Germany

    • Alberto Ardura-Fabregat
    • , Ori Staszewski
    •  & Marco Prinz
  3. Neuroimmunology Unit, Montreal Neurological Institute, Department of Neurology and Neurosurgery, McGill University, Montreal, Quebec, Canada

    • Manon Blain
    • , Luke Healy
    •  & Jack Antel
  4. Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA

    • Loic Lionel Dragin
    •  & Jorge Ivan Alvarez
  5. INSERM, UMR 1064, Nantes, France

    • David A. Laplaud
  6. BIOSS Centre for Biological Signaling Studies, University of Freiburg, Freiburg, Germany

    • Marco Prinz
  7. Broad Institute of MIT and Harvard, Cambridge, MA, USA

    • Francisco J. Quintana

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Contributions

V.R., D.M.B., M.C.T., C.-C.C., A.A.-F., K.A.d.L., C.G.-V., P.H., O.S., M.Bl., L.H., T.N., M.Bo., M.W., L.L.D., D.A.L. and J.I.A. performed in vitro and in vivo experiments, J.A. and M.P. provided unique reagents, discussed and/or interpreted findings, E.C.T. performed bioinformatics, V.R. and F.J.Q. wrote the manuscript and F.J.Q. designed and supervised the study and edited the manuscript.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Francisco J. Quintana.

Extended data figures and tables

  1. Extended Data Fig. 1 Contribution of AHR in CNS resident and infiltrating immune cells during EAE.

    a, qPCR of indicated genes from microglia, splenic macrophages, and astrocytes from control and CX3CR1-AHR mice on day 28 after EAE induction. n = 8 independent samples per group. b, Flow cytometry analysis of AHR expression in microglia, monocytes and astrocytes from Control and CX3CR1-AHR mice 21 days after EAE induction. Thin line depicts isotype control, thick line AHR staining, and numbers indicate percentage of AHR positive cells. Representative of stainings of n = 3 mice per group. c, Spinal cord samples from naive Control and CX3CR1-AHR mice were stained for Iba-1 and DAPI and Iba-1+ microglia/mm2 were determined. n = 5 mice per group. n.s., not significant. d, TUNEL staining in Iba-1+ microglia in spinal cord sections of control and CX3CR1-AHR mice as in c. For the positive control, slides were cooked at 98 °C in citrate buffer during 60 min using a vapour cooker. Solid arrows show TUNEL positive microglia. Representative of n = 5 independent experiments. e, Number of CNS-infiltrating (top) and splenic T cells (bottom), and splenic pro-inflammatory monocytes (bottom) as determined by flow cytometry. n = 5 samples per group for CNS, n = 4 samples per group for spleen. f, Proliferation assay from splenocytes isolated on day 28 of the experiment. n = 4 biologically independent samples per group, representative of two independent experiments. g, Bone marrow chimaera were generated using wild-type mice irradiated as recipients, reconstituted with control or CX3CR1-AHR bone marrow. Recipients of bone marrow were then rested for 3 weeks and thereafter treated with weekly tamoxifen gavages (4 mg) for another 3 weeks; after a total of 6 weeks, EAE was induced and tamoxifen administration continued weekly during EAE. Left, flow cytometry analysis of AHR expression in microglia and monocytes 21 days after EAE induction. Thin line depicts isotype control, thick line denotes AHR staining, and numbers indicate the percentage of AHR-positive cells. Representative of stainings of n= 3 independent mice per group. Right, EAE clinical course in bone marrow chimaera mice. n = 4 mice per group. h, Control and CX3CR1-AHR mice were treated with oral tamoxifen weekly starting from 5 weeks of age. EAE was induced at 8 weeks under continuation of weekly tamoxifen administration. Left, intracellular FACS staining for AHR in microglia and monocytes from at day 21 of EAE. Representative of stainings of n = 3 independent mice per group. Right, clinical course of control and CX3CR1-AHR bone marrow chimaera mice. Data in a, c, eh are mean ± s.e.m. of n = 4 mice per group. P values were determined by two-sided Student’s t-test (a, c, e) or two-way ANOVA (g, h). Source Data.

  2. Extended Data Fig. 2 Topical and molecular regulation of TGFα and VEGF-B.

    a, Ingenuity pathway analysis of differentially regulated pathways in astrocytes from n = 3 control versus CX3CR1-AHR mice per group during EAE. b, Tgfa and Vegfb expression determined by qPCR in microglia from brain, cerebellum and spinal cord 21 days after EAE induction (left). n = 8 mice per group. c, Predicted NF-κB and AHR responsive sites (NREs and XREs, respectively) in Vegfb and Tgfa promoters. d, Microglia were isolated by FACS sorting from control and CX3CR1-AHR mice during EAE. Ex vivo ChIP assay of NF-κB p65 or AHR binding to predicted binding sites in the Vegfb promoter. n = 3 mice per group. Data are representative of two independent experiments. e, Reporter assay using a construct in which the Vegfb promoter controls luciferase expression (pVegfb-Luc). Luciferase activity was measured in HEK293 cells 24 h after transfection with pVegfb-Luc, pTK-Renilla, and plasmids expressing AHR or NF-κB p65. Data are representative of two independent experiments with four biological replicates. f, Ex vivo ChIP assay as in d for AHR binding to the Tgfa promoter. n = 3 mice per group. Representative of two independent experiments. g, Reporter assays using a construct in which the Tgfa promoter controls luciferase expression (pTgfa-Luc). Luciferase activity was measured in HEK293 cells 24 h after transfection with pTgfa-Luc, pTK-Renilla, and plasmids expressing AHR or control. Data are representative of two independent experiments with three biological replicates. Data in a, dg are mean ± s.e.m. P values were determined by one-way ANOVA followed by Tukey’s post-hoc test (b, d, e, f) or two-sided Student’s t-test (g). Source Data.

  3. Extended Data Fig. 3 TGFα and VEGF-B are regulated by AHR in highly purified astrocytes and microglia.

    a, b, Mouse microglia were activated with lipopolysaccharide (LPS) in the presence or absence of the AHR inhibitor CH223191. After 24 h, activation medium was removed and substituted with fresh medium after extensive washes. Then 48 h later, microglia conditioned medium (MCM) was collected and applied to cultures of primary astrocytes. a, Gene expression in microglia 24 h after activation in the presence or absence of CH223191. b, Gene expression in astrocytes after 24 h exposure to MCM. Data are representative of two independent experiments with three biological replicates. c, Representative FACS stainings for CD11b and CD45 in primary astrocyte and microglia cultures. Numbers indicate percentages in respective gate. Data are representative of three independent experiments. d, Representative FACS stainings for GFAP and GLAST in astrocyte cultures as in b. Data are representative of three independent experiments. e, f, qPCR analysis of mRNA expression in astrocyte and microglia cultures. n = 4 independent cultures. Data are representative of two independent experiments with four biological replicates. g, Effect of TGFα and VEGF-B on gene expression in primary astrocytes activated with TNF and IL-1β, determined by pPCR after 24 h. Data are representative of three independent experiments with three biological replicates. h, i, Primary mouse astrocytes were activated with TNF and IL-1β and treated with TGFα or VEGF-B. After 24 h later, culture medium was substituted by fresh medium after extensive washes. Then 48 h later, ACM was added to mouse neurons (h) and oligodendrocytes (i) in culture, and cytotoxicity was determined by quantifying lactate dehydrogenase (LDH) release after 24 h. n = 3 biological replicates. Data are representative of two independent experiments. j, CD11b+Ly6Chi monocyte migration assay performed using ACM from astrocytes activated in the presence of TGFα or VEGF-B. n = 4 biological replicates. Data are representative of two independent experiments. k, qPCR analysis of Nos2 expression in microglia co-cultured with astrocytes activated in the presence of TGFα or VEGF-B. n = 3 biological replicates. Data are representative of two independent experiments. Data in b, ek are mean ± s.e.m. P values were determined by two-sided Student’s t-test (b, e, f) or one-way ANOVA followed by Tukey’s post-hoc test (gk). Source Data.

  4. Extended Data Fig. 4 Phenotypical and functional effects of knockdown of microglial TGFα and VEGF-B.

    a, Quantification of astrocyte numbers in spinal cord sections of knockdown mice. SOX9-positive astrocytes per mm2 were quantified in spinal cord sections of four mice per group. b, IMARIS reconstruction of GFAP+ astrocytes in spinal cord sections as in a, and quantification of dendrite length, branches, volume, terminal points, and segments of n = 4 mice per group. c, d, qPCR analysis of Tgfa and Vegfb expression in sorted CNS-infiltrating inflammatory monocytes (c) and microglia (d) from mice injected with pCD11b-shControl, pCD11b-shTgfa, and pCD11b-shVegfb 7 days after EAE induction. Representative of two independent experiments with three biological replicates. e, qPCR analysis of Erbb1 and Flt1 expression in mice injected with pGFAP-shControl, pGFAP-shErbb1, and pCD11b-shFlt1 7 days after EAE induction. Representative of two independent experiments with three biological replicates. f, Left, flow cytometry analysis of VEGF-B and TGFα expression in microglia from mice injected with pCD11b-shControl, pCD11b-shTgfa, and pCD11b-shVegfb 7 days after EAE induction. Right, quantification of VEGF-B- and TGFα-positive microglia in n = 5 mice per group. Representative of two independent experiments with five biological replicates. g, Left, flow cytometry analysis of FLT-1 and ErbB1 expression in astrocytes from mice injected with pGFAP-shControl, pGFAP-shErbb1, and pCD11b-shFlt1 7 days after EAE induction. Right, quantification of FLT-1 and ErbB1-positive microglia in n = 5 mice per group. Representative of two independent experiments with five biological replicates. h, Naive mice were injected with lysolecithin, VEGF-B, or PBS into the corpus callosum by stereotaxic injection and 6 days later, brains were analysed by myelin staining. Representative of two independent experiments with five biological replicates. Data are mean ± s.e.m. P values were determined one-way ANOVA followed by Tukey’s post-hoc test (ah). Source Data.

  5. Extended Data Fig. 5 Directionality of TGFα and VEGF-B signalling during EAE.

    a, b, EAE development in wild-type mice injected with pGFAP-shControl, pGFAP-shErbb1 and pCD11b-shFlt1 (a), or pCD11b-shControl, pCD11b-shTgfa and pCD11b-shVegfb (b). Clinical course. n = 5 mice per group. Representative of two independent experiments with n = 5 mice per group. c, Left, flow cytometry analysis of TGFα and VEGF-B expression in astrocytes as in a. Right, quantification of cytokine-positive astrocytes. Data are mean ± s.e.m. and P values were determined by one way ANOVA followed by Tukey’s post-hoc test. Representative of two independent experiments with four biological replicates. d, Left, flow cytometry analysis of FLT-1 and ErbB1 expression in microglia as in b. Right, quantification of cell-surface receptor expression of microglia. Data are mean ± s.e.m. and P values were determined by one way ANOVA followed by Tukey’s post-hoc test. Representative of two independent experiments with four biological replicates. Source Data.

  6. Extended Data Fig. 6 Regulation and transcriptional effects of TGFα and VEGF-B during EAE.

    a, b, NanoString analysis of mRNA expression in astrocytes from EAE mice injected with pCD11b-shVegfb or pCD11b-shTgfa (a) and pGFAP-shFlt1 or pGFAP-shErbb1 (b; see also Fig. 2k, l). Fold change in relative expression relative to control as determined by log2(shKD/shControl). shKD, shRNA knockdown. Representative of two independent experiments with pooled RNA isolated from n = 3 mice per group. c, Principal component analysis of gene expression in astrocytes isolated as in a and b. Representative of two independent experiments with pooled RNA isolated from n = 3 mice per group. d, Ingenuity pathway analysis of significantly regulated pathways from astrocytes as in a and b. Representative of two independent experiments with pooled RNA isolated from n = 3 mice per group. e, Left, representative flow cytometry plots depicting NF-κB p65 phosphorylation in wild-type astrocytes stimulated for 15 min with vehicle (top) or TNF or IL-1β (bottom) in the presence of TGFα, VEGF-B, or their combination. Numbers indicate percentage of FITC+ cells. Bar graphs depict quantification of FITC+ cells. Data are mean ± s.e.m. and P values were determined by one way ANOVA followed by Tukey’s post-hoc test. Representative of two independent experiments with four biological replicates. f, Primary mouse astrocytes were exposed to VEGF-B or vehicle and pharmacological blocker of NF-κB activation. RNA was obtained after 18 h and subjected to qPCR analyses for the indicated genes. Data are mean ± s.e.m. and P values were determined by one way ANOVA followed by Tukey’s post-hoc test. Representative of two independent experiments with three biological replicates. g, Primary mouse astrocytes were activated with TNF or IL-1β in the presence of VEGF-B or vehicle, and a pharmacological blocker of NF-κB activation. RNA was obtained after 18 h and subjected to qPCR analyses for the indicated genes. Data are mean ± s.e.m. and P values were determined by one way ANOVA followed by Tukey’s post-hoc test. Representative of two independent experiments with n = 3 biological replicates. Source Data.

  7. Extended Data Fig. 7 Role of AHR in astrocytes and microglia during EAE.

    ac, EAE was induced in control (WT), GfapcreAhrfl/fl (GFAP-AHR), or CX3CR1-AHR EAE mice. Starting from day 7, mice were injected daily intraperitoneally with indoxyl-3-sulfate (I3S), given a tryptophan-depleted diet (TDD), or kept on a control diet. Clinical course of EAE mice under treatment conditions as indicated. Representative of two independent experiments with n = 4 mice per group. df, EAE was induced in wild-type mice, which were treated with lentiviruses to knockdown AHR in astrocytes (pGFAP-shAhr) or microglia (pCD11b-shAhr). A noncoding RNA was used as a control. Flow cytometry quantification of AHR expression in astrocytes and microglia by FACS. d, Representative histograms of n = 4 mice per group. Numbers indicate percentage of AHR-positive cells; thin lines denote isotype control, thick lines denote AHR staining. e, Quantification of AHR-positive astrocytes and microglia as in d. Representative of two independent experiments with four biological replicates. f, EAE mice with knock down of AHR in astrocytes, microglia, or both as in d were subjected to daily I3S injections, TDD, or control diet conditions starting on day 14 after disease induction. Clinical course of n = 4 mice per group. Representative of two independent experiments with n = 4 mice per group. g, Quantification of CNS-infiltrating pro-inflammatory monocytes as determined by FACS at day 28 of EAE. Representative of two independent experiments with three biological replicates. Data are mean ± s.e.m. P values were determined by two-way ANOVA (a, f), or one way ANOVA followed by Tukey’s post-hoc test (e, g). Source Data.

  8. Extended Data Fig. 8 Dietary factors influence mouse and human TGFα and VEGF-B expression.

    a, Ingenuity pathway analysis of NF-κB signalling comparing a TDD to a TDD plus Trp diet in control animals. Colours code for up- and downregulation of individual members in red (up) and blue (down). Normalized reads of two independent samples per group. b, mRNA expression determined by qPCR in from EAE mice as in Fig. 3a. Data are representative of two independent experiments with three replicates. c, Quantification of co-expression of AHR and CD14, VEGF-B and CD14, TGFα and CD14 in immunofluorescence stainings of human white matter brain tissue of NAWM, active, or chronic MS lesions for AHR (left), VEGF-B (middle), or TGFα (right), CD14 (green), and DAPI (blue). Data are representative of n = 12 fields from three distinct MS brains. d, Ratio of VEGF-B to TGFα intensities. Data are the ratio of mean values from Fig. 4e + s.e.m of n = 25 fields. Data in b and d are mean ± s.e.m. P values derived by one-way ANOVA followed by Tukey’s post-hoc test (b, d). Source Data.

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