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Astrocytic A20 ameliorates experimental autoimmune encephalomyelitis by inhibiting NF-κB- and STAT1-dependent chemokine production in astrocytes

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Abstract

Single-nucleotide polymorphisms in the tumor necrosis factor, alpha-induced protein 3 gene, which encodes the ubiquitin-modifying protein A20, are linked to susceptibility to multiple sclerosis (MS), a demyelinating autoimmune disease of the central nervous system (CNS). Since it is unresolved how A20 regulates MS pathogenesis, we examined its function in a murine model of MS, namely experimental autoimmune encephalomyelitis (EAE). Deletion of A20 in neuroectodermal cells (astrocytes, neurons, and oligodendrocytes; Nestin-Cre A20fl/fl mice) or selectively in astrocytes (GFAP-Cre A20fl/fl mice) resulted in more severe EAE as compared to control animals. In Nestin-Cre A20fl/fl and GFAP-Cre A20fl/fl mice demyelination and recruitment of inflammatory leukocytes were increased as compared to A20fl/fl control mice. Importantly, numbers of encephalitogenic CD4+ T cells producing interferon (IFN)-γ, interleukin (IL)-17, and granulocyte–macrophage colony-stimulating factor (GM-CSF), respectively, as well as mRNA production of IFN-γ, IL-17, tumor necrosis factor (TNF), GM-CSF, IL-6, CXCL1, CCL2, and CXCL10 were significantly increased in spinal cords of Nestin-Cre A20fl/fl and GFAP-Cre A20fl/fl mice, respectively. Compared to A20-sufficient astrocytes, A20-deficient astrocytes displayed stronger activation of nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) in response to TNF, IL-17, and GM-CSF, and of signal transducer and activator of transcription 1 (STAT1) upon IFN-γ stimulation. Due to NF-κB and STAT1 hyperactivation, A20-deficient astrocytes produced significantly more chemokines in response to these key encephalitogenic cytokines of autoimmune CD4+ T cells resulting in an amplification of CD4+ T cell recruitment to the CNS. Thus, astrocytic A20 is an important inhibitor of autoimmune-mediated demyelination in the CNS.

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Acknowledgments

The authors thank Elena Fischer, Nadja Schlüter, and Annette Sohnekind for technical assistance. This work was supported by grants of the Deutsche Forschungsgemeinschaft (GRK 1167 to DS; SFB 854/TP5 to DS and MN).

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Authors and Affiliations

  1. Institute of Medical Microbiology, Otto-von-Guericke University Magdeburg, Leipzigerstrasse 44, 39120, Magdeburg, Saxony-Anhalt, Germany

    Xu Wang, Nguyen Thi Xuan, Gopala Nishanth, Sissy Just & Dirk Schlüter

  2. Department of Neuropathology, University Hospital Cologne, 50931, Cologne, Germany

    Martina Deckert

  3. Institute for Molecular Medicine, University Medical Center of the Johannes-Gutenberg University of Mainz, 55131, Mainz, Germany

    Ari Waisman

  4. Institute of Experimental Internal Medicine, Otto-von-Guericke University Magdeburg, 39120, Magdeburg, Germany

    Michael Naumann

  5. Helmholtz Centre for Infection Research, 38124, Braunschweig, Germany

    Dirk Schlüter

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Correspondence to Dirk Schlüter.

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401_2013_1183_MOESM1_ESM.tif

Online Resource 1 Upregulation of A20 in the spinal cord during EAE mRNA transcription of A20 in the spinal cord was evaluated by quantitative RT-PCR in untreated and MOG35-55-immunized C57BL/6 mice at day 15 and 22 p.i (n = 3). Data are expressed as the increase of A20 mRNA of immunized over unimmunized mice normalized to HPRT (mean + SEM * p < 0.05) (TIFF 118 kb)

401_2013_1183_MOESM2_ESM.tif

Online Resource 2 EAE was not aggravated in Nestin-Cre A20wt/wt mice Clinical scores of Nestin-Cre A20wt/wt (n = 12) and A20wt/wt (n = 11) mice after active immunization with MOG35-55 peptide. Data show the mean clinical scores + SEM (TIFF 195 kb)

401_2013_1183_MOESM3_ESM.tif

Online Resource 3 EAE was not aggravated in GFAP-Cre A20wt/wt mice and Synapsin-Cre A20fl/fl mice a WB analysis for A20 expression in FACS-sorted microglia and cultured neurons from GFAP-Cre A20fl/fl and A20fl/fl mice. b Clinical scores of EAE in GFAP-Cre A20wt/wt (n = 5) and A20wt/wt (n = 5) mice induced by MOG35-55-immunization. Data show the mean clinical scores + SEM. c WB analysis for A20 expression in cultured neurons from neuron-restricted A20 deficient (Synapsin-Cre A20fl/fl) and control (A20fl/fl) mice. The right panel shows the relative quantification of A20 normalized to GAPDH. Data show the mean + SEM. d Clinical scores of EAE in Synapsin-Cre A20fl/fl (n = 8) and A20fl/fl (n = 6) mice induced by MOG35-55-immunization. Data show the mean clinical scores + SEM (TIFF 1013 kb)

401_2013_1183_MOESM4_ESM.tif

Online Resource 4 Normal apoptosis of A20-deficient and -sufficient astrocytes after TNF stimulation Astrocytes isolated from GFAP-Cre A20fl/fl and A20fl/fl mice were treated with 20 or 100 ng/ml TNF, respectively, for 24 h. Apoptosis was detected by PI and Annexin staining (TIFF 583 kb)

401_2013_1183_MOESM5_ESM.tif

Online Resource 5 STAT1 expression was upregulated in astrocytes after A20 siRNA treatment a Astrocytes derived from C57BL/6 mice were transfected with siRNA targeting A20 or nonsense siRNA. Sixty hours later, WB analysis was performed on total cell lysates for A20, STAT1 and GAPDH. b Relative quantification of A20 normalized to GAPDH. Data show the mean + SEM. c Relative quantification of STAT1 normalized to GAPDH. Data show the mean + SEM (TIFF 246 kb)

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Wang, X., Deckert, M., Xuan, N.T. et al. Astrocytic A20 ameliorates experimental autoimmune encephalomyelitis by inhibiting NF-κB- and STAT1-dependent chemokine production in astrocytes. Acta Neuropathol 126, 711–724 (2013). https://doi.org/10.1007/s00401-013-1183-9

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