RAR-Related Orphan Receptor Gamma T (RoRγt)-Related Cytokines Play a Role in Neutrophil Infiltration of the Central Nervous System After Subarachnoid Hemorrhage

  • A. P. Coulibaly
  • W. T. Gartman
  • V. Swank
  • J. A. Gomes
  • L. Ruozhuo
  • J. DeBacker
  • J. J. ProvencioEmail author
Original Work



How inflammatory cells are recruited into the central nervous system is a topic of interest in a number of neurological injuries. In aneurysmal subarachnoid hemorrhage (SAH), neutrophil accumulation in the central nervous system 3 days after the hemorrhage is a critical step in the development of delayed cerebral injury (DCI). The mechanism by which neutrophils enter the central nervous system is still unclear.

Methods and Results

To identify human effectors of neutrophil recruitment, cerebrospinal fluid (CSF) samples were taken from a small, selected sample of SAH patients with external ventricular drainage devices (10 patients). Among a battery of CSF cytokines tested 3 days after SAH, five cytokines were associated with poor 90-day outcome (modified Rankin Score 3–6). A parallel study in a mouse model of mild SAH showed elevation in three cytokines in the CNS compared to sham. IL-17 and IL-2 were increased in both patients and the mouse model. IL-17 was investigated further because of its known role in neutrophil recruitment. Inhibition of RAR-Related Orphan Receptor Gamma T, the master transcription factor of IL-17, with the inverse agonist GSK805 suppressed neutrophils entry into the CNS after SAH compared to control. Using an IL-17 reporter mouse, we investigated the source of IL-17 and found that myeloid cells were a common IL-17-producing cell type in the meninges after SAH, suggesting an autocrine role for neutrophil recruitment.


Taken together, IL-17 appears to be in important factor in the recruitment of neutrophils into the meninges after SAH and could be an important target for therapies to ameliorate DCI.


Subarachnoid hemorrhage Neuroinflammation Neutrophils IL-17 Delayed cerebral injury 



Bovine serum albumin


Central nervous system


Cerebrospinal fluid


Dulbecco’s modified Eagle medium


Delayed cerebral injury




External ventricular drain


Green fluorescent protein




Modified Rankin Scale


Multiple sclerosis


Phosphate-buffered saline


RAR-related orphan receptor gamma t


Subarachnoid hemorrhage


Tumor necrosis factor


Author’s contributions

APC and WTG conceived the mouse experiments, synthesized data and wrote the manuscript, VS and JD collected samples, analyzed human data samples and revised the manuscript, JAG collected human samples, supervised human outcome data and revised manuscript, LR developed techniques, performed mouse experiments, and revised the manuscript, JJP conceived of experiments, supervised the entire project, reviewed and revised the manuscript.

Source of support

This work was funded by The Aneurysm and AVM Foundation (JJP and JAG), NIH 1RO1NS0749971 and K08 NS 051350 (JJP).

Conflict of interest

No author reports conflicts of interest for this work.

Ethical approval/informed consent

Human subject study was approved by the Cleveland Clinic Institutional Review Board and was conducted over parts of 2013 and 2014. All patients were consented for participation. All animal experiments were done with the approval of the University of Virginia Animal Care and Use Committee.

Supplementary material

12028_2019_871_MOESM1_ESM.pdf (637 kb)
Supplementary material 1 (PDF 636 kb)


  1. 1.
    Suarez JI. Diagnosis and management of subarachnoid hemorrhage. Continuum (Minneap Minn). 2015;21(5 Neurocritical Care):1263–87.Google Scholar
  2. 2.
    Diringer MN, Bleck TP, Claude Hemphill J 3rd, et al. Critical care management of patients following aneurysmal subarachnoid hemorrhage: recommendations from the Neurocritical Care Society’s Multidisciplinary Consensus Conference. Neurocrit Care. 2011;15(2):211–40.CrossRefGoogle Scholar
  3. 3.
    Suarez JI, Tarr RW, Selman WR. Aneurysmal subarachnoid hemorrhage. N Engl J Med. 2006;354(4):387–96.CrossRefGoogle Scholar
  4. 4.
    Al-Khindi T, Macdonald RL, Schweizer TA. Cognitive and functional outcome after aneurysmal subarachnoid hemorrhage. Stroke. 2010;41(8):e519–36.CrossRefGoogle Scholar
  5. 5.
    Wong GK, Lam S, Ngai K, et al. Evaluation of cognitive impairment by the Montreal cognitive assessment in patients with aneurysmal subarachnoid haemorrhage: prevalence, risk factors and correlations with 3 month outcomes. J Neurol Neurosurg Psychiatry. 2012;83(11):1112–7.CrossRefGoogle Scholar
  6. 6.
    Macdonald RL, Higashida RT, Keller E, et al. Randomised trial of clazosentan, an endothelin receptor antagonist, in patients with aneurysmal subarachnoid hemorrhage undergoing surgical clipping (CONSCIOUS-2). Acta Neurochir Suppl. 2013;115:27–31.PubMedGoogle Scholar
  7. 7.
    Stuart D, Christian R, Uschmann H, Palokas M. Effectiveness of intrathecal nicardipine on cerebral vasospasm in non-traumatic subarachnoid hemorrhage: a systematic review. JBI Database System Rev Implement Rep. 2018;16(10):2013–26.CrossRefGoogle Scholar
  8. 8.
    Dhar R, Diringer MN. The burden of the systemic inflammatory response predicts vasospasm and outcome after subarachnoid hemorrhage. Neurocrit Care. 2008;8(3):404–12.CrossRefGoogle Scholar
  9. 9.
    Provencio JJ, Vora N. Subarachnoid hemorrhage and inflammation: bench to bedside and back. Semin Neurol. 2005;25(4):435–44.CrossRefGoogle Scholar
  10. 10.
    Provencio JJ, Swank V, Lu H, et al. Neutrophil depletion after subarachnoid hemorrhage improves memory via NMDA receptors. Brain Behav Immun. 2016;54:233–42.CrossRefGoogle Scholar
  11. 11.
    Provencio JJ, Fu X, Siu A, Rasmussen PA, Hazen SL, Ransohoff RM. CSF neutrophils are implicated in the development of vasospasm in subarachnoid hemorrhage. Neurocrit Care. 2010;12(2):244–51.CrossRefGoogle Scholar
  12. 12.
    Gaetani P, Tartara F, Pignatti P, Tancioni F, Rodriguez y Baena R, De Benedetti F. Cisternal CSF levels of cytokines after subarachnoid hemorrhage. Neurol Res. 1998;20(4):337–42.CrossRefGoogle Scholar
  13. 13.
    Hirashima Y, Nakamura S, Endo S, Kuwayama N, Naruse Y, Takaku A. Elevation of platelet activating factor, inflammatory cytokines, and coagulation factors in the internal jugular vein of patients with subarachnoid hemorrhage. Neurochem Res. 1997;22(10):1249–55.CrossRefGoogle Scholar
  14. 14.
    Zeiler FA, Thelin EP, Czosnyka M, Hutchinson PJ, Menon DK, Helmy A. Cerebrospinal fluid and microdialysis cytokines in aneurysmal subarachnoid hemorrhage: a scoping systematic review. Front Neurol. 2017;8:379.CrossRefGoogle Scholar
  15. 15.
    Polin RS, Bavbek M, Shaffrey ME, et al. Detection of soluble E-selectin, ICAM-1, VCAM-1, and L-selectin in the cerebrospinal fluid of patients after subarachnoid hemorrhage. J Neurosurg. 1998;89(4):559–67.CrossRefGoogle Scholar
  16. 16.
    Savarraj JPJ, Parsha K, Hergenroeder GW, et al. Systematic model of peripheral inflammation after subarachnoid hemorrhage. Neurology. 2017;88(16):1535–45.CrossRefGoogle Scholar
  17. 17.
    Provencio JJ, Altay T, Smithason S, Moore SK, Ransohoff RM. Depletion of Ly6G/C(+) cells ameliorates delayed cerebral vasospasm in subarachnoid hemorrhage. J Neuroimmunol. 2011;232(1–2):94–100.CrossRefGoogle Scholar
  18. 18.
    Zhang R, Tian A, Wang J, Shen X, Qi G, Tang Y. miR26a modulates Th17/T reg balance in the EAE model of multiple sclerosis by targeting IL6. Neuromolecular Med. 2015;17(1):24–34.CrossRefGoogle Scholar
  19. 19.
    Wang B, Tang Y, Sun X, et al. Increased IL-6 expression on THP-1 by IL-34 stimulation up-regulated rheumatoid arthritis Th17 cells. Clin Rheumatol. 2018;37:127–37.CrossRefGoogle Scholar
  20. 20.
    Shoda H, Nagafuchi Y, Tsuchida Y, et al. Increased serum concentrations of IL-1 beta, IL-21 and Th17 cells in overweight patients with rheumatoid arthritis. Arthritis Res Ther. 2017;19(1):111.CrossRefGoogle Scholar
  21. 21.
    Ferretti S, Bonneau O, Dubois GR, Jones CE, Trifilieff A. IL-17, produced by lymphocytes and neutrophils, is necessary for lipopolysaccharide-induced airway neutrophilia: IL-15 as a possible trigger. J Immunol. 2003;170(4):2106–12.CrossRefGoogle Scholar
  22. 22.
    Li L, Huang L, Vergis AL, et al. IL-17 produced by neutrophils regulates IFN-gamma-mediated neutrophil migration in mouse kidney ischemia-reperfusion injury. J Clin Invest. 2010;120(1):331–42.CrossRefGoogle Scholar
  23. 23.
    Katayama M, Ohmura K, Yukawa N, et al. Neutrophils are essential as a source of IL-17 in the effector phase of arthritis. PLoS ONE. 2013;8(5):e62231.CrossRefGoogle Scholar
  24. 24.
    Lin AM, Rubin CJ, Khandpur R, et al. Mast cells and neutrophils release IL-17 through extracellular trap formation in psoriasis. J Immunol. 2011;187(1):490–500.CrossRefGoogle Scholar
  25. 25.
    Chaudhry SR, Guresir E, Vatter H, et al. Aneurysmal subarachnoid hemorrhage lead to systemic upregulation of IL-23/IL-17 inflammatory axis. Cytokine. 2017;97:96–103.CrossRefGoogle Scholar
  26. 26.
    Altay T, Smithason S, Volokh N, Rasmussen PA, Ransohoff RM, Provencio JJ. A novel method for subarachnoid hemorrhage to induce vasospasm in mice. J Neurosci Methods. 2009;183(2):136–40.CrossRefGoogle Scholar
  27. 27.
    Xiao S, Yosef N, Yang J, et al. Small-molecule RORgammat antagonists inhibit T helper 17 cell transcriptional network by divergent mechanisms. Immunity. 2014;40(4):477–89.CrossRefGoogle Scholar
  28. 28.
    Gadani SP, Smirnov I, Smith AT, Overall CC, Kipnis J. Characterization of meningeal type 2 innate lymphocytes and their response to CNS injury. J Exp Med. 2017;214(2):285–96.CrossRefGoogle Scholar
  29. 29.
    Mrdjen D, Pavlovic A, Hartmann FJ, et al. High-dimensional single-cell mapping of central nervous system immune cells reveals distinct myeloid subsets in health, aging, and disease. Immunity. 2018;48(3):599.CrossRefGoogle Scholar
  30. 30.
    Deniset JF, Surewaard BG, Lee WY, Kubes P. Splenic Ly6G(high) mature and Ly6G(int) immature neutrophils contribute to eradication of S. pneumoniae. J Exp Med. 2017;214(5):1333–50.CrossRefGoogle Scholar
  31. 31.
    Savarraj J, Parsha K, Hergenroeder G, et al. Early brain injury associated with systemic inflammation after subarachnoid hemorrhage. Neurocrit Care. 2018;28(2):203–11.CrossRefGoogle Scholar
  32. 32.
    Ji KA, Eu MY, Kang SH, Gwag BJ, Jou I, Joe EH. Differential neutrophil infiltration contributes to regional differences in brain inflammation in the substantia nigra pars compacta and cortex. Glia. 2008;56(10):1039–47.CrossRefGoogle Scholar
  33. 33.
    Barone FC, Hillegass LM, Price WJ, et al. Polymorphonuclear leukocyte infiltration into cerebral focal ischemic tissue: myeloperoxidase activity assay and histologic verification. J Neurosci Res. 1991;29(3):336–45.CrossRefGoogle Scholar
  34. 34.
    Roth TL, Nayak D, Atanasijevic T, Koretsky AP, Latour LL, McGavern DB. Transcranial amelioration of inflammation and cell death after brain injury. Nature. 2014;505(7482):223–8.CrossRefGoogle Scholar
  35. 35.
    Corps KN, Roth TL, McGavern DB. Inflammation and neuroprotection in traumatic brain injury. JAMA Neurol. 2015;72(3):355–62.CrossRefGoogle Scholar
  36. 36.
    Sarkar S, Fox DA. Targeting IL-17 and Th17 cells in rheumatoid arthritis. Rheum Dis Clin North Am. 2010;36(2):345–66.CrossRefGoogle Scholar
  37. 37.
    Kostic M, Dzopalic T, Zivanovic S, et al. IL-17 and glutamate excitotoxicity in the pathogenesis of multiple sclerosis. Scand J Immunol. 2014;79(3):181–6.CrossRefGoogle Scholar
  38. 38.
    Hu S, He W, Du X, et al. IL-17 production of neutrophils enhances antibacteria ability but promotes arthritis development during mycobacterium tuberculosis infection. EBioMedicine. 2017;23:88–99.CrossRefGoogle Scholar
  39. 39.
    Coffelt SB, Kersten K, Doornebal CW, et al. IL-17-producing gammadelta T cells and neutrophils conspire to promote breast cancer metastasis. Nature. 2015;522(7556):345–8.CrossRefGoogle Scholar
  40. 40.
    Taylor PR, Roy S, Leal SM Jr, et al. Activation of neutrophils by autocrine IL-17A-IL-17RC interactions during fungal infection is regulated by IL-6, IL-23, RORgammat and dectin-2. Nat Immunol. 2014;15(2):143–51.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature and Neurocritical Care Society 2019

Authors and Affiliations

  1. 1.Department of Neurology and NeuroscienceUniversity of VirginiaCharlottesvilleUSA
  2. 2.University of North CarolinaChapel HillUSA
  3. 3.NeuroscienceCleveland Clinic Lerner Research InstituteClevelandUSA
  4. 4.Cerebrovascular CenterCleveland ClinicClevelandUSA
  5. 5.Department of NeurologyChinese PLA General HospitalBeijingChina
  6. 6.Department of AnesthesiologyUniversity of TorontoOntarioUSA

Personalised recommendations