Peroxiredoxin family proteins are key initiators of post-ischemic inflammation in the brain

Journal name:
Nature Medicine
Volume:
18,
Pages:
911–917
Year published:
DOI:
doi:10.1038/nm.2749
Received
Accepted
Published online

Abstract

Post-ischemic inflammation is an essential step in the progression of brain ischemia-reperfusion injury. However, the mechanism that activates infiltrating macrophages in the ischemic brain remains to be clarified. Here we demonstrate that peroxiredoxin (Prx) family proteins released extracellularly from necrotic brain cells induce expression of inflammatory cytokines including interleukin-23 in macrophages through activation of Toll-like receptor 2 (TLR2) and TLR4, thereby promoting neural cell death, even though intracellular Prxs have been shown to be neuroprotective. The extracellular release of Prxs in the ischemic core occurred 12 h after stroke onset, and neutralization of extracellular Prxs with antibodies suppressed inflammatory cytokine expression and infarct volume growth. In contrast, high mobility group box 1 (HMGB1), a well-known damage-associated molecular pattern molecule, was released before Prx and had a limited role in post-ischemic macrophage activation. We thus propose that extracellular Prxs are previously unknown danger signals in the ischemic brain and that its blocking agents are potent neuroprotective tools.

At a glance

Figures

  1. Peroxiredoxins in brain lysate are potent inducers of IL-23 expression.
    Figure 1: Peroxiredoxins in brain lysate are potent inducers of IL-23 expression.

    (a) Time course of inflammatory cytokine expression in BMDCs from WT or Myd88−/− mice cultured with sham-operated or ischemic brain lysate (n = 6 for each). (b) IL-23 p19 mRNA levels in BMDCs 1 h after stimulation with brain lysate that had been treated with heat (98 °C, 10 min), pronase or DNase I (n = 3 for each). (c) IL-23 p19 mRNA levels in BMDCs 1 h after stimulation with the indicated sucrose-gradient fractions of the brain lysate (representative data from one of five independent experiments). (d) IL-23 p19 mRNA levels in BMDCs 1 h after stimulation with the indicated recombinant GST-fusion proteins (n = 3 for each) that are detected in sucrose-gradient fractions 2 and 3 by LC-MS. (e) Western blot analysis of the indicated proteins in the sucrose-gradient fraction of the brain lysate. (f,g) mRNA levels of inflammatory cytokines (f) and IL-23 protein levels (g) induced in BMDC by the addition of recombinant Prxs or GST protein or lipopolysaccharide (LPS, 100 ng ml−1) (n = 3 for each). (h) IL-23 p19–inducing activity of each sucrose gradient fraction after immunoprecipitation using control IgG or the Prx-specific antibody mixture. The depletion of Prxs was confirmed by western blot analysis (left; representative data from two independent experiments). Each mRNA expression level indicates relative expression compared to that in untreated BMDCs. ***P < 0.001 versus brain lysate (one-way analysis of variance (ANOVA) with Dunnett's correction; the error bars represent s.e.m.).

  2. Extracellular peroxiredoxin colocalizes with infiltrating macrophages in the ischemic brain.
    Figure 2: Extracellular peroxiredoxin colocalizes with infiltrating macrophages in the ischemic brain.

    (a) Time course of mRNA levels of Prx family proteins in ischemic brain (n = 3 for each). Each value indicates relative expression level compared to Hprt1 mRNA. (b) Time course of immunohistochemical staining of Prx6 in ischemic brain tissue at the indicated time points (scale bars, 50 μm). (c) Immunohistochemical staining of day-1 ischemic brain tissue (scale bar, 50 μm). (d) Double immunohistochemical staining for Prx6 and TUNEL in the infarct core on day 1 (scale bar, 10 μm). (e,f) Double immunohistochemical staining for Prx6 and F4/80 in the infarct core on day 1 (scale bar, 10 μm). The graph in f shows a quantification of fluorescence intensity along the yellow arrow in left panel. The images were captured by using conventional fluorescence microscopy (b,c) or confocal laser microscopy (df). The error bars represent s.e.m.

  3. Peroxiredoxins induce IL-23 expression via TLR2 and TLR4.
    Figure 3: Peroxiredoxins induce IL-23 expression via TLR2 and TLR4.

    (a) IL-23 p19 mRNA levels in WT, TLR2-deficient, TLR4-deficient or TLR2 and TLR4 double-deficient BMDC 1 h after stimulation with 1 μM recombinant Prx proteins (n = 4 for each), shown relative to those in untreated BMDCs. (b) The mRNA levels of inflammatory cytokines in the infiltrating immune cells on day 1 after stroke onset (n = 7 for each). Each value indicates relative expression compared to that in sham-operated mice. (c) The ratio and the absolute number of IL-17+ T cells on day 3 after stroke onset (n = 8 for WT and n = 6 for other samples). (d) Infarct volume (bottom) measured by MAP2 immunostaining of brain sections on day 4 after stroke onset in chimeric mice (top; scale bars, 1 mm). Tlr2−/− BM right arrow WT means transfer of Tlr2−/− bone marrow cells into WT mice. The number of mice is shown on each bar. *P < 0.05, **P < 0.01, ***P < 0.001 versus WT mice (b,c) and WT bone marrow–transferred WT mice (d) (one-way ANOVA with Dunnett's correction; the error bars represent s.e.m.).

  4. Extracellular release of Prxs contributes to the initiation of post-ischemic inflammation.
    Figure 4: Extracellular release of Prxs contributes to the initiation of post-ischemic inflammation.

    (a,b) Infarct volume on day 4 after stroke onset (a) and neurological scores on days 1 and 3 after stroke onset (b) of mice treated with control IgG (500 μg per mouse), a mixture of antibodies specific for Prx1, Prx2, Prx5 and Prx6 (anti-Prxs; 500 μg per mouse), HMGB1-specific antibody (anti-HMGB1; 200 μg per mouse), or both Prx-specific antibodies and HMGB1-specific antibody immediately after stroke onset (scale bars, 1 mm). i.v., intravenous; MCAO, middle cerebral artery occlusion. (c,d) Infarct volume on day 4 after stroke onset (c) and neurological scores (d) on days 1 and 3 after stroke onset of TLR2 and TLR4 double-deficient mice treated with control IgG or the Prx-specific antibody mixture immediately after stroke onset (n = 7 for each) (scale bars, 1 mm). (eg) mRNA levels of inflammatory cytokines in brain infiltrating immune cells on day 1 after stroke onset (n = 11 for control IgG, n = 7 for Prx-specific antibodies, and n = 8 for HMGB1-specific antibody) (e), the ratio and the absolute number of IL-17+ T cells on day 3 after stroke onset (n = 8 for each) (f) and the absolute number of TUNEL+ cells in the peri-infarct area on day 4 after stroke onset (scale bars, 50 μm; n = 8 for each) (g) in the mice treated with control IgG, Prx-specific antibody mixture or HMGB1-specific antibody immediately after stroke onset. Each mRNA expression level indicates relative expression compared to that in sham-operated mice. The number of mice is shown on each bar (a,c). *P < 0.05, **P < 0.01, ***P < 0.001 versus control IgG-administered mice (one-way ANOVA with Dunnett's correction (a,b,e) or two-sided Student's t test (c,d,f,g); the error bars represent s.e.m.).

  5. Neutralization of extracellular Prxs is neuroprotective within 12 h after stroke onset.
    Figure 5: Neutralization of extracellular Prxs is neuroprotective within 12 h after stroke onset.

    (a,b) Infarct volume on day 4 after stroke onset (a) and neurological scores on days 1 and 3 after stroke onset (b) of mice treated with control IgG, Prx-specific antibody mixture or HMGB1-specific antibody 6 h after stroke onset (scale bars, 1 mm). (c,d) Infarct volume on day 4 after stroke onset (c) and neurological scores on days 1 and 3 after stroke onset (d) of mice treated with control IgG or Prx-specific antibody mixture 12 h after stroke onset (scale bars, 1 mm). (e,f) Infarct volume on day 4 after stroke onset (e) and neurological scores on days 1 and 3 after stroke onset (f) of mice treated with control IgG or Prx-specific antibody mixture 24 h after stroke onset (scale bars, 1 mm). The number of mice is shown on each bar (a,c,e). *P < 0.05 versus control IgG–administered mice (one-way ANOVA with Dunnett's correction (a,b) or two-sided Student's t test (cf); the error bars represent s.e.m.).

  6. The conserved region of peroxiredoxins was essential for IL-23-inducing activity and the increase in infarct size.
    Figure 6: The conserved region of peroxiredoxins was essential for IL-23-inducing activity and the increase in infarct size.

    (a) IL-23 p19–inducing activities of C-terminal deletion mutants of Prx5 (n = 3 for each). The number of amino acid residues contained in each GST-fusion Prx5 peptide is shown on the x axis. (b) IL-23 p19–inducing activities of GST-fusion Prx1, Prx5, and Prx6 peptides (n = 3 for each). The number of amino acid residues contained in each Prx1, Prx5 and Prx6 peptide is shown on the x axis (GST-Prx147–70, GST-Prx542–67 and GST-Prx641–65 peptides contained the α2 helix region; GST-Prx171–94, GST-Prx568–90 and GST-Prx666–93 peptides contained the β4 sheet and α3 helix regions). IL-23 p19 mRNA levels were detected by means of quantitative RT-PCR in BMDCs 1 h after stimulation with 1 μM recombinant proteins and are shown relative to those of untreated BMDCs. (c,d) Infarct volume on day 4 after stroke onset (scale bars, 1 mm) (c) and neurological scores (d) on days 1 and 3 after stroke onset of mice treated with control IgG, a Prx5- and Prx6-specific antibody mixture (anti-Prx5/Prx6) or a Prx568–90- and Prx666–93-specific (common α3 helix and β4 sheet region) antibody mixture (anti-Prx568–90/Prx666–93) immediately after the induction of brain ischemia (300 μg per mouse). The number of mice is shown on each bar in c. *P < 0.05, **P < 0.01, ***P < 0.001 versus GST-Prx51–162 (a) and control IgG-treated mice (c,d) (one-way ANOVA with Dunnett's correction; the error bars represent s.e.m.).

References

  1. Moskowitz, M.A., Lo, E.H. & Iadecola, C. The science of stroke: mechanisms in search of treatments. Neuron 67, 181198 (2010).
  2. Lo, E.H. Degeneration and repair in central nervous system disease. Nat. Med. 16, 12051209 (2010).
  3. Ooboshi, H. et al. Postischemic gene transfer of interleukin-10 protects against both focal and global brain ischemia. Circulation 111, 913919 (2005).
  4. Macrez, R. et al. Stroke and the immune system: from pathophysiology to new therapeutic strategies. Lancet Neurol. 10, 471480 (2011).
  5. Iadecola, C. & Anrather, J. The immunology of stroke: from mechanisms to translation. Nat. Med. 17, 796808 (2011).
  6. Eltzschig, H.K. & Eckle, T. Ischemia and reperfusion—from mechanism to translation. Nat. Med. 17, 13911401 (2011).
  7. Hurn, P.D. et al. T- and B-cell–deficient mice with experimental stroke have reduced lesion size and inflammation. J. Cereb. Blood Flow Metab. 27, 17981805 (2007).
  8. Liesz, A. et al. Inhibition of lymphocyte trafficking shields the brain against deleterious neuroinflammation after stroke. Brain 134, 704720 (2011).
  9. Yilmaz, G., Arumugam, T.V., Stokes, K.Y. & Granger, D.N. Role of T lymphocytes and interferon-gamma in ischemic stroke. Circulation 113, 21052112 (2006).
  10. Ren, X. et al. Regulatory B cells limit CNS inflammation and neurologic deficits in murine experimental stroke. J. Neurosci. 31, 85568563 (2011).
  11. Shichita, T. et al. Pivotal role of cerebral interleukin-17-producing γδT cells in the delayed phase of ischemic brain injury. Nat. Med. 15, 946950 (2009).
  12. Konoeda, F. et al. Therapeutic effect of IL-12/23 and their signaling pathway blockade on brain ischemia model. Biochem. Biophys. Res. Commun. 402, 500506 (2010).
  13. Tang, S.C. et al. Pivotal role for neuronal Toll-like receptors in ischemic brain injury and functional deficits. Proc. Natl. Acad. Sci. USA 104, 1379813803 (2007).
  14. Chen, C.J. et al. Identification of a key pathway required for the sterile inflammatory response triggered by dying cells. Nat. Med. 13, 851856 (2007).
  15. Yanai, H. et al. HMGB proteins function as universal sentinels for nucleic-acid-mediated innate immune responses. Nature 462, 99103 (2009).
  16. Marsh, B.J., Williams-Karnesky, R.L. & Stenzel-Poore, M.P. Toll-like receptor signaling in endogenous neuroprotection and stroke. Neuroscience 158, 10071020 (2009).
  17. Stewart, C.R. et al. CD36 ligands promote sterile inflammation through assembly of a Toll-like receptor 4 and 6 heterodimer. Nat. Immunol. 11, 155161 (2010).
  18. Zhang, Q. et al. Circulating mitochondrial DAMPs cause inflammatory responses to injury. Nature 464, 104107 (2010).
  19. Rivest, S. Regulation of innate immune responses in the brain. Nat. Rev. Immunol. 9, 429439 (2009).
  20. Zhang, J. et al. Anti–high mobility group box-1 monoclonal antibody protects the blood-brain barrier from ischemia-induced disruption in rats. Stroke 42, 14201428 (2011).
  21. Kim, J.B. et al. HMGB1, a novel cytokine-like mediator linking acute neuronal death and delayed neuroinflammation in the postischemic brain. J. Neurosci. 26, 64136421 (2006).
  22. Yang, Q.W. et al. HMBG1 mediates ischemia-reperfusion injury by TRIF-adaptor independent Toll-like receptor 4 signaling. J. Cereb. Blood Flow Metab. 31, 593605 (2011).
  23. Hayakawa, K., Qiu, J. & Lo, E.H. Biphasic actions of HMGB1 signaling in inflammation and recovery after stroke. Ann. NY Acad. Sci. 1207, 5057 (2010).
  24. Riddell, J.R., Wang, X.Y., Minderman, H. & Gollnick, S.O. Peroxiredoxin 1 stimulates secretion of proinflammatory cytokines by binding to TLR4. J. Immunol. 184, 10221030 (2010).
  25. Chesterman, E.S. et al. Investigation of Prx1 protein expression provides evidence for conservation of cardiac-specific posttranscriptional regulation in vertebrates. Dev. Dyn. 222, 459470 (2001).
  26. Patenaude, A., Murthy, M.R. & Mirault, M.E. Emerging roles of thioredoxin cycle enzymes in the central nervous system. Cell. Mol. Life Sci. 62, 10631080 (2005).
  27. Hu, X. et al. Peroxiredoxin-2 protects against 6-hydroxydopamine–induced dopaminergic neurodegeneration via attenuation of the apoptosis signal-regulating kinase (ASK1) signaling cascade. J. Neurosci. 31, 247261 (2011).
  28. Rashidian, J. et al. Essential role of cytoplasmic cdk5 and Prx2 in multiple ischemic injury models, in vivo. J. Neurosci. 29, 1249712505 (2009).
  29. Wood, Z.A., Schröder, E., Robin Harris, J. & Poole, L.B. Structure, mechanism and regulation of peroxiredoxins. Trends Biochem. Sci. 28, 3240 (2003).
  30. Seo, M.S. et al. Identification of a new type of mammalian peroxiredoxin that forms an intramolecular disulfide as a reaction intermediate. J. Biol. Chem. 275, 2034620354 (2000).
  31. Toohey, J.I. Sulfhydryl dependence in primary explant hematopoietic cells. Inhibition of growth in vitro with vitamin B12 compounds. Proc. Natl. Acad. Sci. USA 72, 7377 (1975).
  32. Chou, J.L. et al. Proteomic investigation of a neural substrate intimately related to brain death. Proteomics 11, 239248 (2011).
  33. Dayon, L. et al. Brain extracellular fluid protein changes in acute stroke patients. J. Proteome Res. 10, 10431051 (2011).
  34. Jin, M.H. et al. Characterization of neural cell types expressing peroxiredoxins in mouse brain. Neurosci. Lett. 381, 252257 (2005).
  35. Brea, D. et al. Toll-like receptors 2 and 4 in ischemic stroke: outcome and therapeutic values. J. Cereb. Blood Flow Metab. 31, 14241431 (2011).
  36. Hirotsu, S. et al. Crystal structure of a multifunctional 2-Cys peroxiredoxin heme-binding protein 23kDa/proliferation-associated gene product. Proc. Natl. Acad. Sci. USA 96, 1233312338 (1999).
  37. Declercq, J.P. et al. Crystal structure of human peroxiredoxin 5, a novel type of mammalian peroxiredoxin at 1.5 A resolution. J. Mol. Biol. 311, 751759 (2001).
  38. Choi, H.J. et al. Crystal structure of a novel human peroxidase enzyme at 2.0 A resolution. Nat. Struct. Biol. 5, 400406 (1998).
  39. Chen, G.Y. & Nuñez, G. Sterile inflammation: sensing and reacting to damage. Nat. Rev. Immunol. 10, 826837 (2010).
  40. Yu, M. et al. HMGB1 signals through Toll-like receptor (TLR) 4 and TLR2. Shock 26, 174179 (2006).
  41. Triantafilou, M. et al. Membrane sorting of Toll-like receptor (TLR)-2/6 and TLR2/1 heterodimers at the cell surface determines heterotypic associations with CD36 and intracellular targeting. J. Biol. Chem. 281, 3100231011 (2006).
  42. Yi, H. et al. Pattern recognition scavenger receptor SRA/CD204 down-regulates Toll-like receptor 4 signaling–dependent CD8 T-cell activation. Blood 113, 58195828 (2009).
  43. Akashi-Takamura, S. & Miyake, K. TLR accessory molecules. Curr. Opin. Immunol. 20, 420425 (2008).
  44. Eismann, T. et al. Peroxiredoxin-6 protects against mitochondrial dysfunction and liver injury during ischemia-reperfusion in mice. Am. J. Physiol. Gastrointest. Liver Physiol. 296, G266G274 (2009).
  45. Liu, K. et al. Anti-high mobility group box 1 monoclonal antibody ameliorates brain infarction induced by transient ischemia in rats. FASEB J. 21, 39043916 (2007).
  46. Qiu, J. et al. Early release of HMGB-1 from neurons after the onset of brain ischemia. J. Cereb. Blood Flow Metab. 28, 927938 (2008).
  47. Sugimori, H. et al. Krypton laser-induced photothrombotic distal middle cerebral artery occlusion without craniectomy in mice. Brain Res Brain Res. Protoc. 13, 189196 (2004).

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Author information

Affiliations

  1. Department of Microbiology and Immunology, School of Medicine, Keio University, Tokyo, Japan.

    • Takashi Shichita,
    • Eiichi Hasegawa,
    • Akihiro Kimura,
    • Rimpei Morita,
    • Ryota Sakaguchi,
    • Ichiro Takada,
    • Takashi Sekiya &
    • Akihiko Yoshimura
  2. Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency, Tokyo, Japan.

    • Takashi Shichita
  3. Department of Medicine and Clinical Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.

    • Takashi Shichita &
    • Takanari Kitazono
  4. Department of Internal Medicine, Fukuoka Dental College Medical and Dental Hospital, Fukuoka, Japan.

    • Hiroaki Ooboshi
  5. Faculty of Medicine, University of Tsukuba, Tsukuba, Japan.

    • Toru Yanagawa &
    • Tetsuro Ishii
  6. Department of Pharmacology, Okayama University Graduate School of Medicine, Okayama, Japan.

    • Hideo Takahashi,
    • Shuji Mori &
    • Masahiro Nishibori
  7. Division of Microbiology, Nihon University School of Medicine, Tokyo, Japan.

    • Kazumichi Kuroda
  8. Laboratory of Host Defense, WPI Immunology Frontier Research Center, Osaka University, Osaka, Japan.

    • Shizuo Akira
  9. Division of Infectious Genetics, Institute of Medical Science, University of Tokyo, Tokyo, Japan.

    • Kensuke Miyake
  10. Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, Tokyo, Japan.

    • Akihiko Yoshimura

Contributions

T. Shichita designed and performed experiments, analyzed data and wrote the manuscript; E.H. and A.K. performed TLR-deficient mouse analysis; R.M., R.S. and T. Sekiya participated in data analysis and discussion; I.T. provided specific input on protein analysis; H.O. and T.K. provided technical advice about experimental design; T.Y. and T.I. provided crucial input on Prx1's functions; H.T., S.M. and M.N. provided the HMGB1-specific antibody and crucial input on HMGB1; K.K. provided specific input regarding LC-MS analysis; K.M. and S.A. provided TLR2 and/or TLR4-deficient mice; A.Y. initiated and directed the entire study, designed experiments and wrote the manuscript.

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The authors declare no competing financial interests.

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