Abstract
The molecular mechanisms that link the sympathetic stress response and inflammation remain obscure. Here we found that the transcription factor Nr4a1 regulated the production of norepinephrine (NE) in macrophages and thereby limited experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. Lack of Nr4a1 in myeloid cells led to enhanced NE production, accelerated infiltration of leukocytes into the central nervous system (CNS) and disease exacerbation in vivo. In contrast, myeloid-specific deletion of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, protected mice against EAE. Furthermore, we found that Nr4a1 repressed autocrine NE production in macrophages by recruiting the corepressor CoREST to the Th promoter. Our data reveal a new role for macrophages in neuroinflammation and identify Nr4a1 as a key regulator of catecholamine production by macrophages.
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Acknowledgements
We thank D. Metzger (Institut Génétique Biologie Moléculaire Cellulaire) and H. Ichinose (Tokyo Institute of Technology) for Nr4a1fl/fl mice; K. Ley for discussions; A. Rao for guidance in composing the manuscript; A. Crotti for guidance on microglia culture; and D. Yoakum for assistance with mouse colony management. Supported by the American Heart Association (13SDG17060117 to I.S. and 12SDG12070005 to R.N.H.), the La Jolla Institute Board of Directors (R.N.H.), Fondation Leducq (M.U.K.), the Sigrid Juselius Foundation (M.U.K.), the Academy of Finland (M.U.K.), the Pacific Northwest Udall Center (P50-NS062684 to M.D.) and the US National Institutes of Health (R01 DK091183-21 to C.K.G. and R01 HL118765 to C.C.H.).
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I.S., R.N.H. and H.S. designed, performed and analyzed the experiments; G.C., H.N.N. and R.T. designed and preformed the experiments; G.T., R.T., A.B.B., Z.M., S.T., J.M., A.B., M.U.K., S.L.-W.-S. and A.R. performed the experiments; S.S.-A., M.D., G.D.T., A.B.-O., C.K.G., H.B. and C.C.H. designed the experiments; and I.S., R.N.H., H.S. and C.C.H. wrote the manuscript.
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Integrated supplementary information
Supplementary Figure 1 EAE induction.
(a) Schematic of autoreactive 2D2 T cell stimulation, adoptive transfer and EAE disease induction in WT and Nr4a1–/– mice. (b) Flow cytometry analysis of TNFα and IFN-γ production in expanded and stimulated autoreactive 2D2 T cells prior to transfer. (c) CD64 and MerTK expression on macrophages and monocytes gated as in Main Fig. 1b.
Supplementary Figure 2 Loss of Nr4a1 leads to accelerated and exacerbated EAE.
(a) EAE progression following transfer of 0.5, 1 or 2 million autoreactive 2D2 T cells. Note that regardless of the number of injected cells, all mice eventually develop disease while Nr4a1–/– mice develop accelerated and worsened disease compared to their WT counterparts. p<0.01, p<0.001, p<0.001, respectively. (b) EAE progression following transfer of 1 million 2D2 T cells, monitored till the end point; note that WT mice eventually exhibit full disease progression, but in a significantly longer time compared to Nr4a1–/– mice. p<0.001. (c) Change in body mass percentage in WT and Nr4a1–/– mice with EAE progression following transfer of 0.5, 1 or 2 million 2D2 T cells. p<0.01, p<0.05, p<0.01, respectively. (d) EAE disease progression in WT and Nr4a1–/– mice after active immunization with MOG in complete Freund’s adjuvant. p<0.001. (e) Change in body mass percentage following (left) transfer of 1 million 2D2 cells into Nr4a1fl/fl (WT) or LysM-Cre;Nr4a1fl/fl (Nr4a1ΔLysM) and (right) transfer of 0.5 million 2D2 cells into Nr4a1fl/fl (WT) or Csfr-Cre;Nr4a1fl/fl (Nr4a1ΔCsfr) recipients. p<0.05, p<0.001, respectively. (f) One million 2D2 cells derived on Nr4a1–/– or WT backgrounds were transferred into WT mice and disease progression was scored. p>0.05. Two way-ANOVA test; error bars, s.e.m. (n = 5/group in each experiment).
Supplementary Figure 4 Early T cell infiltration, but similar blood-brain-barrier permeability, in Nr4a1–/– mice.
(a-b) Quantification of 2D2 T cell infiltration on day 4 (a) or 7 (b), as described in Main Fig. 2f. (c) Evans Blue concentration in the brain following i.v. injection; 3 mice in each group; unpaired Student’s t-test (p>0.05); error bars, s.e.m.
Supplementary Figure 5 Early microglial activation in Nr4a1–/– mice at the induction of EAE.
(a) Quantification of microglia dendricity (top) as a measure of microglia activation; representative images (bottom) on day 7 of disease onset as described in Main Fig. 2f. (b-c) A representative flow cytometry plot (b), and quantification (c) of MHC class II and CD44 expression in microglia analyzed by flow cytometry upon EAE onset. Four mice per group were analyzed.
Supplementary Figure 6 Greater infiltration of 2D2 T cells and microglial activation in the brain of Nr4a1–/– mice than in Nr4a1+/+ mice at 7 d after transfer.
Confocal imaging of DsRed 2D2 T cell infiltrate (red), Cx3cr1-GFP+ microglia/ monocytes (green) and vasculature (blue) in the brain (cerebral cortex) of WT and Nr4a1–/– mice 7 days after the transfer of 1 million 2D2 T cell transfer.
Supplementary Figure 7 NE-producing macrophages have an essential role in EAE.
(a,b) The β1 (atenolol) (a) and the β2 (butaxamine) (b) blockers were administered (250 μg i.p. twice a day) and compared to untreated (UN) WT or Nr4a1–/– mice after the transfer of 1 million 2D2 cells; EAE was scored over 12 days (n = 5/group). (c) TH mRNA in BMM from WT or DDfs mice. Expression relative to WT is shown. (d) To confirm TH deletion in ThΔLysM mice, the cells were freshly isolated from the CNS at day 15 post EAE induction and analyzed via flow cytometry; 3 animals were analyzed in each group; gating scheme as in Fig. 1b (middle lower panel). (e) NE blood concentration in WT and ThΔLysM mice at day 15 post EAE induction, 3 animals in each group. *** p<0.001, ** p<0.01, * p<0.05 (unpaired Student’s t-test and 2-way Anova test); error bars, s.e.m.
Supplementary Figure 8 Nr4a1 is a negative regulator of TH in vitro.
(a) Nr4a1 mRNA expression in BMM following treatment with IFN-γ or with NE. (b, c) Flow cytometry of BMM shows Nr4a1-GFP induction following IFN-γ (b) or NE (c) treatment. A representative of 2 experiments is shown. (d) NE concentration in the conditioned media of BMM, untreated (CTL) or treated with IFN-γ, with or without 100 μM AMPT, a TH inhibitor, or 100 μM 6-OHDA, a toxin inducing chemical sympathectomy, analyzed by ELISA, 4 replicates in each group. (e) TH-luciferase reporter assay in macrophages. RAW or BMM cells were transfected with β-gal construct and TH-luciferase reporter construct; luciferase activity was measured and normalized to β-gal activity and to the activity in cells without IFN-γ treatment. BMM were derived from WT or Nr4a1–/– mice. RAW cells were transfected with either control or Nr4a1 siRNA. Three replicates in each group, a representative of 2 experiments shown. *** p<0.001, ** p<0.01, * p<0.05 (unpaired Student’s t-test and 2-way Anova test); error bars, s.e.m.
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Supplementary Figures 1–8 (PDF 1427 kb)
Autoreactive T cell infiltration into CNS
Two million DsRed-2D2 T cells were transferred into WT and Nr4a1–/– mice on CX3CR1-GFP background, and Ly6G-APC was injected just before imaging to visualize neutrophils and blood vessels (blue). The imaging was performed in the naïve animal. Representative results of three separate experiments are showing in vivo confocal imaging of the spinal cord adjacent to the posterior spinal vein (PSV). (MOV 25393 kb)
Autoreactive T cell infiltration into CNS
Two million DsRed-2D2 T cells were transferred into WT and Nr4a1–/– mice on CX3CR1-GFP background, and Ly6G-APC was injected just before imaging to visualize neutrophils and blood vessels (blue). The imaging was performed in the animal at score 1.5 (limp tail) o Representative results of three separate experiments are showing in vivo confocal imaging of the spinal cord adjacent to the posterior spinal vein (PSV).f the EAE. (MOV 15293 kb)
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Shaked, I., Hanna, R., Shaked, H. et al. Transcription factor Nr4a1 couples sympathetic and inflammatory cues in CNS-recruited macrophages to limit neuroinflammation. Nat Immunol 16, 1228–1234 (2015). https://doi.org/10.1038/ni.3321
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DOI: https://doi.org/10.1038/ni.3321
