Commensal gut bacteria impact the host immune system and can influence disease processes in several organs, including the brain. However, it remains unclear whether the microbiota has an impact on the outcome of acute brain injury. Here we show that antibiotic-induced alterations in the intestinal flora reduce ischemic brain injury in mice, an effect transmissible by fecal transplants. Intestinal dysbiosis alters immune homeostasis in the small intestine, leading to an increase in regulatory T cells and a reduction in interleukin (IL)-17–positive γδ T cells through altered dendritic cell activity. Dysbiosis suppresses trafficking of effector T cells from the gut to the leptomeninges after stroke. Additionally, IL-10 and IL-17 are required for the neuroprotection afforded by intestinal dysbiosis. The findings reveal a previously unrecognized gut-brain axis and an impact of the intestinal flora and meningeal IL-17+ γδ T cells on ischemic injury.
Subscribe to Journal
Get full journal access for 1 year
only $4.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Henninger, N., Kumar, R. & Fisher, M. Acute ischemic stroke therapy. Expert Rev. Cardiovasc. Ther. 8, 1389–1398 (2010).
Iadecola, C. & Anrather, J. The immunology of stroke: from mechanisms to translation. Nat. Med. 17, 796–808 (2011).
Macrez, R. et al. Stroke and the immune system: from pathophysiology to new therapeutic strategies. Lancet Neurol. 10, 471–480 (2011).
Mazmanian, S.K., Liu, C.H., Tzianabos, A.O. & Kasper, D.L. An immunomodulatory molecule of symbiotic bacteria directs maturation of the host immune system. Cell 122, 107–118 (2005).
Prinz, I., Silva-Santos, B. & Pennington, D.J. Functional development of γδ T cells. Eur. J. Immunol. 43, 1988–1994 (2013).
Shichita, T. et al. Pivotal role of cerebral interleukin-17–producing γδ T cells in the delayed phase of ischemic brain injury. Nat. Med. 15, 946–950 (2009).
Gelderblom, M. et al. Neutralization of the IL-17 axis diminishes neutrophil invasion and protects from ischemic stroke. Blood 120, 3793–3802 (2012).
Liesz, A., Hu, X., Kleinschnitz, C. & Offner, H. Functional role of regulatory lymphocytes in stroke: facts and controversies. Stroke 46, 1422–1430 (2015).
Liesz, A. et al. Regulatory T cells are key cerebroprotective immunomodulators in acute experimental stroke. Nat. Med. 15, 192–199 (2009).
Stubbe, T. et al. Regulatory T cells accumulate and proliferate in the ischemic hemisphere for up to 30 days after MCAO. J. Cereb. Blood Flow Metab. 33, 37–47 (2013).
Li, P. et al. Adoptive regulatory T cell therapy protects against cerebral ischemia. Ann. Neurol. 74, 458–471 (2013).
Chaudhry, A. et al. CD4+ regulatory T cells control TH17 responses in a Stat3-dependent manner. Science 326, 986–991 (2009).
Huber, S. et al. TH17 cells express interleukin-10 receptor and are controlled by Foxp3− and Foxp3+ regulatory CD4+ T cells in an interleukin-10–dependent manner. Immunity 34, 554–565 (2011).
Park, S.-G. et al. T regulatory cells maintain intestinal homeostasis by suppressing γδ T cells. Immunity 33, 791–803 (2010).
Cho, S. et al. The class B scavenger receptor CD36 mediates free radical production and tissue injury in cerebral ischemia. J. Neurosci. 25, 2504–2512 (2005).
Kunz, A. et al. Neurovascular protection by ischemic tolerance: role of nitric oxide and reactive oxygen species. J. Neurosci. 27, 7083–7093 (2007).
Braniste, V. et al. The gut microbiota influences blood-brain barrier permeability in mice. Sci. Transl. Med. 6, 263ra158 (2014).
Hu, X., Li, P. & Chen, J. Pro: regulatory T cells are protective in ischemic stroke. Stroke 44, e85–e86 (2013).
Round, J.L. & Mazmanian, S.K. The gut microbiota shapes intestinal immune responses during health and disease. Nat. Rev. Immunol. 9, 313–323 (2009).
Nishio, J. & Honda, K. Immunoregulation by the gut microbiota. Cell. Mol. Life Sci. 69, 3635–3650 (2012).
Justicia, C. et al. Neutrophil infiltration increases matrix metalloproteinase–9 in the ischemic brain after occlusion-reperfusion of the middle cerebral artery in rats. J. Cereb. Blood Flow Metab. 23, 1430–1440 (2003).
Stowe, A.M. et al. Neutrophil elastase and neurovascular injury following focal stroke and reperfusion. Neurobiol. Dis. 35, 82–90 (2009).
Engelhardt, B. & Ransohoff, R.M. The ins and outs of T lymphocyte trafficking to the CNS: anatomical sites and molecular mechanisms. Trends Immunol. 26, 485–495 (2005).
Morton, A.M. et al. Endoscopic photoconversion reveals unexpectedly broad leukocyte trafficking to and from the gut. Proc. Natl. Acad. Sci. USA 111, 6696–6701 (2014).
Nowotschin, S. & Hadjantonakis, A.-K. Use of KikGR, a photoconvertible green-to-red fluorescent protein, for cell labeling and lineage analysis in ES cells and mouse embryos. BMC Dev. Biol. 9, 49 (2009).
Coombes, J.L. et al. A functionally specialized population of mucosal CD103+ DCs induces Foxp3+ regulatory T cells via a TGF-β– and retinoic acid–dependent mechanism. J. Exp. Med. 204, 1757–1764 (2007).
Scott, C.L., Aumeunier, A.M. & Mowat, A.M. Intestinal CD103+ dendritic cells: master regulators of tolerance? Trends Immunol. 32, 412–419 (2011).
Ochoa-Repáraz, J. et al. A polysaccharide from the human commensal Bacteroides fragilis protects against CNS demyelinating disease. Mucosal Immunol. 3, 487–495 (2010).
Rescigno, M. et al. Dendritic cells express tight-junction proteins and penetrate gut epithelial monolayers to sample bacteria. Nat. Immunol. 2, 361–367 (2001).
Niess, J.H. et al. CX3CR1-mediated dendritic cell access to the intestinal lumen and bacterial clearance. Science 307, 254–258 (2005).
Josefowicz, S.Z., Lu, L.-F. & Rudensky, A.Y. Regulatory T cells: mechanisms of differentiation and function. Annu. Rev. Immunol. 30, 531–564 (2012).
Louveau, A. et al. Structural and functional features of central nervous system lymphatic vessels. Nature 523, 337–341 (2015).
Kleinschnitz, C. & Wiendl, H. Con: regulatory T cells are protective in ischemic stroke. Stroke 44, e87–e88 (2013).
Kleinschnitz, C. et al. Regulatory T cells are strong promoters of acute ischemic stroke in mice by inducing dysfunction of the cerebral microvasculature. Blood 121, 679–691 (2013).
Roth, T.L. et al. Transcranial amelioration of inflammation and cell death after brain injury. Nature 505, 223–228 (2014).
Pérez-de-Puig, I. et al. Neutrophil recruitment to the brain in mouse and human ischemic stroke. Acta Neuropathol. 129, 239–257 (2015).
Kleinschnitz, C. et al. Early detrimental T cell effects in experimental cerebral ischemia are neither related to adaptive immunity nor to thrombus formation. Blood 115, 3835–3842 (2010).
Li, G.-Z. et al. Expression of interleukin-17 in ischemic brain tissue. Scand. J. Immunol. 62, 481–486 (2005).
Kostulas, N., Pelidou, S.H., Kivisäkk, P., Kostulas, V. & Link, H. Increased IL-1β, IL-8 and IL-17 mRNA expression in blood mononuclear cells observed in a prospective ischemic stroke study. Stroke 30, 2174–2179 (1999).
Erbel, C. et al. Expression of IL-17A in human atherosclerotic lesions is associated with increased inflammation and plaque vulnerability. Basic Res. Cardiol. 106, 125–134 (2011).
Abraham, C. & Cho, J. Interleukin-23–TH17 pathways and inflammatory bowel disease. Inflamm. Bowel Dis. 15, 1090–1100 (2009).
Keller, J.J. et al. Increased risk of stroke among patients with Crohn's disease: a population-based matched cohort study. Int. J. Colorectal Dis. 30, 645–653 (2015).
Singh, S., Kullo, I.J., Pardi, D.S. & Loftus, E.V. Jr. Epidemiology, risk factors and management of cardiovascular diseases in IBD. Nat. Rev. Gastroenterol. Hepatol. 12, 26–35 (2015).
Kilkenny, C., Browne, W., Cuthill, I.C., Emerson, M. & Altman, D.G. Animal research: reporting in vivo experiments—the ARRIVE guidelines. J. Cereb. Blood Flow Metab. 31, 991–993 (2011).
Ivanov, I.I. et al. Induction of intestinal TH17 cells by segmented filamentous bacteria. Cell 139, 485–498 (2009).
Snel, J. et al. Comparison of 16S rRNA sequences of segmented filamentous bacteria isolated from mice, rats and chickens, and proposal of 'Candidatus arthromitus'. Int. J. Syst. Bacteriol. 45, 780–782 (1995).
Barman, M. et al. Enteric salmonellosis disrupts the microbial ecology of the murine gastrointestinal tract. Infect. Immun. 76, 907–915 (2008).
Benson, A.K. et al. Individuality in gut microbiota composition is a complex polygenic trait shaped by multiple environmental and host genetic factors. Proc. Natl. Acad. Sci. USA 107, 18933–18938 (2010).
Ubeda, C. et al. Familial transmission rather than defective innate immunity shapes the distinct intestinal microbiota of TLR-deficient mice. J. Exp. Med. 209, 1445–1456 (2012).
Caricilli, A.M. et al. Gut microbiota is a key modulator of insulin resistance in Tlr2-knockout mice. PLoS Biol. 9, e1001212 (2011).
Jackman, K., Kunz, A. & Iadecola, C. Modeling focal cerebral ischemia in vivo. Methods Mol. Biol. 793, 195–209 (2011).
Lin, T.N., He, Y.Y., Wu, G., Khan, M. & Hsu, C.Y. Effect of brain edema on infarct volume in a focal cerebral ischemia model in rats. Stroke 24, 117–121 (1993).
Bouët, V. et al. Sensorimotor and cognitive deficits after transient middle cerebral artery occlusion in the mouse. Exp. Neurol. 203, 555–567 (2007).
Yagi, S. & Costanzo, R.M. Grafting the olfactory epithelium to the olfactory bulb. Am. J. Rhinol. Allergy 23, 239–243 (2009).
Jackman, K. et al. Progranulin deficiency promotes post-ischemic blood-brain barrier disruption. J. Neurosci. 33, 19579–19589 (2013).
Schloss, P.D. et al. Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl. Environ. Microbiol. 75, 7537–7541 (2009).
Buffie, C.G. et al. Precision microbiome reconstitution restores bile acid–mediated resistance to Clostridium difficile. Nature 517, 205–208 (2015).
Sheneman, L., Evans, J. & Foster, J.A. Clearcut: a fast implementation of relaxed neighbor joining. Bioinformatics 22, 2823–2824 (2006).
Garcia-Bonilla, L., Racchumi, G., Murphy, M., Anrather, J. & Iadecola, C. Endothelial CD36 contributes to postischemic brain injury by promoting neutrophil activation via CSF3. J. Neurosci. 35, 14783–14793 (2015).
Pino, P.A. & Cardona, A.E. Isolation of brain and spinal cord mononuclear cells using Percoll gradients. J. Vis. Exp. 48, 2348 (2011).
Roederer, M. Spectral compensation for flow cytometry: visualization artifacts, limitations and caveats. Cytometry 45, 194–205 (2001).
Liaw, A. & Wiener, M. Classification and regression by randomForest. R News 2, 18–22 (2002).
J.A. is the recipient of the Finbar and Marianne Kenny Research Scholarship. Parts of the study were supported by the US National Institutes of Health (NIH) grants NS081179 (J.A.) and NS34179 (C.I. and J.A.), the Feil Family Foundation (C.I.) and the Swiss National Science Foundation for Grants in Biology and Medicine (P3SMP3 148367; C.B.). We thank A.-K. Hadjantonakis (Memorial Sloan Kettering Cancer Center) for helpful discussions on the use of the KikGR33 mice.
The authors declare no competing financial interests.
About this article
Cite this article
Benakis, C., Brea, D., Caballero, S. et al. Commensal microbiota affects ischemic stroke outcome by regulating intestinal γδ T cells. Nat Med 22, 516–523 (2016). https://doi.org/10.1038/nm.4068
Brain, Behavior, and Immunity (2021)
Journal of Neuroimmunology (2021)
International Journal of Molecular Sciences (2021)
Nano Research (2021)
Journal of Clinical Medicine (2021)