Regulatory T cells are key cerebroprotective immunomodulators in acute experimental stroke

Abstract

Systemic and local inflammatory processes have a key, mainly detrimental role in the pathophysiology of ischemic stroke. Currently, little is known about endogenous counterregulatory immune mechanisms. We examined the role of the key immunomodulators CD4+CD25+ forkhead box P3 (Foxp3)+ regulatory T lymphocytes (Treg cells), after experimental brain ischemia. Depletion of Treg cells profoundly increased delayed brain damage and deteriorated functional outcome. Absence of Treg cells augmented postischemic activation of resident and invading inflammatory cells including microglia and T cells, the main sources of deleterious cerebral tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), respectively. Early antagonization of TNF-α and delayed neutralization of IFN-γ prevented infarct growth in Treg cell–depleted mice. Intracerebral interleukin-10 (IL-10) substitution abrogated the cytokine overexpression after Treg cell depletion and prevented secondary infarct growth, whereas transfer of IL-10–deficient Treg cells in an adoptive transfer model was ineffective. In conclusion, Treg cells are major cerebroprotective modulators of postischemic inflammatory brain damage targeting multiple inflammatory pathways. IL-10 signaling is essential for their immunomodulatory effect.

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Figure 1: Treg cell depletion exacerbates ischemic lesion size and behavioral outcome.
Figure 2: Proinflammatory cytokine expression is elevated in the ischemic brain after Treg cell depletion, and their intracerebral antagonization reduces lesion size.
Figure 3: Treg cells reduce the early invasion of neutrophils into the brain and the activation of invading T cells.
Figure 4: Microglia are activated after ischemia, are the main source of cerebral TNF-α and are more abundant in brains of Treg cell–depleted mice.
Figure 5: Blood cytokine concentrations after MCAO.
Figure 6: Treg cell–derived IL-10 is the main mediator of the Treg cells' cerebroprotective effect.

References

  1. 1

    Donnan, G.A., Fisher, M., Macleod, M. & Davis, S.M. Stroke. Lancet 371, 1612–1623 (2008).

  2. 2

    Lo, E.H., Dalkara, T. & Moskowitz, M.A. Mechanisms, challenges and opportunities in stroke. Nat. Rev. Neurosci. 4, 399–415 (2003).

  3. 3

    Dirnagl, U. Inflammation in stroke: the good, the bad, and the unknown. Ernst Schering Res. Found. Workshop. 47, 87–99 (2004).

  4. 4

    Wang, Q., Tang, X.N. & Yenari, M.A. The inflammatory response in stroke. J. Neuroimmunol. 184, 53–68 (2007).

  5. 5

    Arumugam, T.V., Granger, D.N. & Mattson, M.P. Stroke and T cells. Neuromolecular Med. 7, 229–242 (2005).

  6. 6

    Perera, M.N. et al. Inflammation following stroke. J. Clin. Neurosci. 13, 1–8 (2006).

  7. 7

    Meisel, C., Schwab, J.M., Prass, K., Meisel, A. & Dirnagl, U. Central nervous system injury-induced immune deficiency syndrome. Nat. Rev. Neurosci. 6, 775–786 (2005).

  8. 8

    Gee, J.M., Kalil, A., Shea, C. & Becker, K.J. Lymphocytes: potential mediators of postischemic injury and neuroprotection. Stroke 38, 783–788 (2007).

  9. 9

    McGeachy, M.J., Stephens, L.A. & Anderton, S.M. Natural recovery and protection from autoimmune encephalomyelitis: contribution of CD4+CD25+ regulatory cells within the central nervous system. J. Immunol. 175, 3025–3032 (2005).

  10. 10

    Suri-Payer, E. & Fritzsching, B. Regulatory T cells in experimental autoimmune disease. Springer Semin. Immunopathol. 28, 3–16 (2006).

  11. 11

    Sakaguchi, S. et al. Foxp3+ CD25+ CD4+ natural regulatory T cells in dominant self-tolerance and autoimmune disease. Immunol. Rev. 212, 8–27 (2006).

  12. 12

    O'Connor, R.A. & Anderton, S.M. Foxp3+ regulatory T cells in the control of experimental CNS autoimmune disease. J. Neuroimmunol. 193, 1–11 (2008).

  13. 13

    O'Garra, A. & Vieira, P. Regulatory T cells and mechanisms of immune system control. Nat. Med. 10, 801–805 (2004).

  14. 14

    Tang, Q. & Bluestone, J.A. The Foxp3+ regulatory T cell: a jack of all trades, master of regulation. Nat. Immunol. 9, 239–244 (2008).

  15. 15

    Spera, P.A., Ellison, J.A., Feuerstein, G.Z. & Barone, F.C. IL-10 reduces rat brain injury following focal stroke. Neurosci. Lett. 251, 189–192 (1998).

  16. 16

    Gee, J.M., Kalil, A., Thullbery, M. & Becker, K.J. Induction of immunologic tolerance to myelin basic protein prevents central nervous system autoimmunity and improves outcome after stroke. Stroke 39, 1575–1582 (2008).

  17. 17

    Offner, H. et al. Splenic atrophy in experimental stroke is accompanied by increased regulatory T cells and circulating macrophages. J. Immunol. 176, 6523–6531 (2006).

  18. 18

    McNeill, A., Spittle, E. & Backstrom, B.T. Partial depletion of CD69-expressing natural regulatory T cells with the anti-CD25 monoclonal antibody PC61. Scand. J. Immunol. 65, 63–69 (2007).

  19. 19

    Schallert, T., Fleming, S.M., Leasure, J.L., Tillerson, J.L. & Bland, S.T. CNS plasticity and assessment of forelimb sensorimotor outcome in unilateral rat models of stroke, cortical ablation, parkinsonism and spinal cord injury. Neuropharmacology 39, 777–787 (2000).

  20. 20

    Zhang, L. et al. A test for detecting long-term sensorimotor dysfunction in the mouse after focal cerebral ischemia. J. Neurosci. Methods 117, 207–214 (2002).

  21. 21

    Barone, F.C. et al. Tumor necrosis factor-α. A mediator of focal ischemic brain injury. Stroke 28, 1233–1244 (1997).

  22. 22

    Yilmaz, G., Arumugam, T.V., Stokes, K.Y. & Granger, D.N. Role of T lymphocytes and interferon-γ in ischemic stroke. Circulation 113, 2105–2112 (2006).

  23. 23

    Lambertsen, K.L. et al. A role for interferon-γ in focal cerebral ischemia in mice. J. Neuropathol. Exp. Neurol. 63, 942–955 (2004).

  24. 24

    Lambertsen, K.L., Meldgaard, M., Ladeby, R. & Finsen, B. A quantitative study of microglial-macrophage synthesis of tumor necrosis factor during acute and late focal cerebral ischemia in mice. J. Cereb. Blood Flow Metab. 25, 119–135 (2005).

  25. 25

    Rothwell, N.J. & Luheshi, G.N. Interleukin 1 in the brain: biology, pathology and therapeutic target. Trends Neurosci. 23, 618–625 (2000).

  26. 26

    Gregersen, R., Lambertsen, K. & Finsen, B. Microglia and macrophages are the major source of tumor necrosis factor in permanent middle cerebral artery occlusion in mice. J. Cereb. Blood Flow Metab. 20, 53–65 (2000).

  27. 27

    Ito, D., Tanaka, K., Suzuki, S., Dembo, T. & Fukuuchi, Y. Enhanced expression of Iba1, ionized calcium-binding adapter molecule 1, after transient focal cerebral ischemia in rat brain. Stroke 32, 1208–1215 (2001).

  28. 28

    Offner, H. et al. Experimental stroke induces massive, rapid activation of the peripheral immune system. J. Cereb. Blood Flow Metab. 26, 654–665 (2006).

  29. 29

    Grilli, M. et al. Interleukin-10 modulates neuronal threshold of vulnerability to ischaemic damage. Eur. J. Neurosci. 12, 2265–2272 (2000).

  30. 30

    del Zoppo, G.J., Becker, K.J. & Hallenbeck, J.M. Inflammation after stroke: is it harmful? Arch. Neurol. 58, 669–672 (2001).

  31. 31

    Dirnagl, U., Iadecola, C. & Moskowitz, M.A. Pathobiology of ischaemic stroke: an integrated view. Trends Neurosci. 22, 391–397 (1999).

  32. 32

    del Zoppo, G. et al. Inflammation and stroke: putative role for cytokines, adhesion molecules and iNOS in brain response to ischemia. Brain Pathol. 10, 95–112 (2000).

  33. 33

    Chamorro, A., Urra, X. & Planas, A.M. Infection after acute ischemic stroke: a manifestation of brain-induced immunodepression. Stroke 38, 1097–1103 (2007).

  34. 34

    Prass, K. et al. Stroke-induced immunodeficiency promotes spontaneous bacterial infections and is mediated by sympathetic activation reversal by poststroke T helper cell type 1–like immunostimulation. J. Exp. Med. 198, 725–736 (2003).

  35. 35

    Planas, A.M., Gorina, R. & Chamorro, A. Signalling pathways mediating inflammatory responses in brain ischaemia. Biochem. Soc. Trans. 34, 1267–1270 (2006).

  36. 36

    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, 1798–1805 (2007).

  37. 37

    Schroeter, M. & Jander, S. T-cell cytokines in injury-induced neural damage and repair. Neuromolecular Med. 7, 183–195 (2005).

  38. 38

    Wajant, H., Pfizenmaier, K. & Scheurich, P. Tumor necrosis factor signaling. Cell Death Differ. 10, 45–65 (2003).

  39. 39

    Baird, A.E. The forgotten lymphocyte: immunity and stroke. Circulation 113, 2035–2036 (2006).

  40. 40

    Campanella, M., Sciorati, C., Tarozzo, G. & Beltramo, M. Flow cytometric analysis of inflammatory cells in ischemic rat brain. Stroke 33, 586–592 (2002).

  41. 41

    Fisson, S. et al. Continuous activation of autoreactive CD4+ CD25+ regulatory T cells in the steady state. J. Exp. Med. 198, 737–746 (2003).

  42. 42

    Frenkel, D. et al. Neuroprotection by IL-10–producing MOG CD4+ T cells following ischemic stroke. J. Neurol. Sci. 233, 125–132 (2005).

  43. 43

    Strle, K. et al. Interleukin-10 in the brain. Crit. Rev. Immunol. 21, 427–449 (2001).

  44. 44

    Vignali, D.A., Collison, L.W. & Workman, C.J. How regulatory T cells work. Nat. Rev. Immunol. 8, 523–532 (2008).

  45. 45

    O'Garra, A., Vieira, P.L., Vieira, P. & Goldfeld, A.E. IL-10–producing and naturally occurring CD4+ Tregs: limiting collateral damage. J. Clin. Invest. 114, 1372–1378 (2004).

  46. 46

    Vogel, J., Mobius, C. & Kuschinsky, W. Early delineation of ischemic tissue in rat brain cryosections by high-contrast staining. Stroke 30, 1134–1141 (1999).

  47. 47

    Schallert, T., Hernandez, T.D. & Barth, T.M. Recovery of function after brain damage: severe and chronic disruption by diazepam. Brain Res. 379, 104–111 (1986).

  48. 48

    Steiner, G.E., Ecker, R.C., Kramer, G., Stockenhuber, F. & Marberger, M.J. Automated data acquisition by confocal laser scanning microscopy and image analysis of triple stained immunofluorescent leukocytes in tissue. J. Immunol. Methods 237, 39–50 (2000).

  49. 49

    Blais, V. & Rivest, S. Effects of TNF-α and IFN-γ on nitric oxide–induced neurotoxicity in the mouse brain. J. Immunol. 172, 7043–7052 (2004).

  50. 50

    Nadeau, S. & Rivest, S. Role of microglial-derived tumor necrosis factor in mediating CD14 transcription and nuclear factor κ B activity in the brain during endotoxemia. J. Neurosci. 20, 3456–3468 (2000).

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Acknowledgements

This study was supported by grants from the Else-Kröner Fresenius Stiftung and the Ministerium für Wissenschaft und Kultur Baden-Württemberg to R.V. We would like to thank H. Bürgers and D. Stefan for excellent technical assistance and I. Galani (German Cancer Research Center, Heidelberg) for providing the Rag2−/− mice.

Author information

A.L. designed and performed experiments, analyzed data and wrote the manuscript; H.D. performed experiments and analyzed data; E.S.-P. provided crucial input on Treg cell function and contributed to experimental design and manuscript writing; C.V. designed experiments and contributed to manuscript writing; C.S. designed experiments and analyzed data; S.R. performed experiments and analyzed data; T.G. provided specific input to flow cytometric analysis and contributed to manuscript writing; R.V. initiated and directed the entire study, designed experiments, analyzed data and wrote the manuscript.

Correspondence to Roland Veltkamp.

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Liesz, A., Suri-Payer, E., Veltkamp, C. et al. Regulatory T cells are key cerebroprotective immunomodulators in acute experimental stroke. Nat Med 15, 192–199 (2009). https://doi.org/10.1038/nm.1927

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