Journal of Molecular Neuroscience

, Volume 61, Issue 3, pp 385–395 | Cite as

NLRP3 is Required for Complement-Mediated Caspase-1 and IL-1beta Activation in ICH

  • Sheng-Tao Yao
  • Fang Cao
  • Jia-Lin Chen
  • Wei Chen
  • Rui-Ming Fan
  • Gang Li
  • You-Chao Zeng
  • Song Jiao
  • Xiang-Ping Xia
  • Chong Han
  • Qi-Shan Ran


Complement-mediated inflammation plays a vital role in intracerebral hemorrhage (ICH), implicating pro-inflammatory factor interleukin-1beta (IL-1β) secretion. Brain samples and contralateral hemiencephalon were all collected and detected by Western blot. NLRP3 expression was located by dual immunofluorescence staining at 1, 3, and 5 days post-ICH. Brain water content was examined post-ICH. The neural deficit scores were evaluated by observers blindly. ILs were detected by ELISA. SiRNAs targeting NLRP3 (siNLRP3), siASC, and siControl were injected to inhibit NLRP3 function. To test the complement activation via Nod-like receptor (NLR) family pyrin domain-containing 3 (NLRP3), normal rabbit complement (NRC) was injected with lipopolysaccharide (LPS) to facilitate the complement function. As a result, complement 3a (C3a) and complement 5a (C5a) were upregulated during the ICH-induced neuroinflammation, and ablation of C3 attenuates ICH-induced IL-1β release. Though the LPS rescues the neuroinflammation in the ICH model, C3 deficiency attenuates the LPS-induced inflammatory effect. The NLRP3 inflammasome was activated after ICH and was located in the microglial cell of the mouse brain, which exhibits a time-dependent manner. However, the number of NLRP3/Iba-1 dual-labeled cells in the C3−/− group is less than that in the WT group in each time course, respectively. IL-1β and IL-18 released in perihematoma tissue, caspase-1-p20, brain water content, and behavioral outcomes were attenuated in the siNLRP3 and siASC groups than in the siControl and ICH groups. We also found that 5% of complement supplement enhances ICH-induced IL-1β release, while NLRP3 and ASC inhibition attenuates it. In conclusion, complement-induced ICH neuroinflammation depended on NLRP3 activation, which facilities LPS- and ICH-induced neuroinflammation, and NLRP3 is required for ICH-induced inflammation.


Intracerebral hemorrhage Complement, NLRP3 inflammasome Neuroinflammation IL-1β 



This work was supported by the National Natural Science Foundation of China (81660211) and the Major Program of Science and Technology Foundation of Zunyi (Guizhou) Technology Bureau.

Author Contributions

STY was involved in performing the majority of the laboratory-based work and writing of the manuscript. JLC, WC, and RMF helped with laboratory work concerning control tissues and were involved in the writing of the manuscript. GL, YCZ, SJ, XPX, and CH were involved in collecting data, statistics, and troubleshooting. STY and PW were involved in conceptualizing and planning of the presented work.

Compliance with Ethical Standards

All animal studies followed the guidelines outlined in the Guide for the Care and Use of Laboratory Animals from the National Institute of Health and were approved by the Zunyi Hospital of Zunyi Animal Welfare Committee.

Conflict of Interest

The authors declare that they have no conflict of interest.


  1. Block ML, Zecca L, Hong JS (2007) Microglia-mediated neurotoxicity: uncovering the molecular mechanisms. Nat Rev Neurosci 8:57–69. doi: 10.1038/nrn2038 CrossRefPubMedGoogle Scholar
  2. Bonnardeaux A, Pichette V (2003) Complement dysregulation in haemolytic uraemic syndrome. Lancet 362:1514–1515. doi: 10.1016/S0140-6736(03)14777-0 CrossRefPubMedGoogle Scholar
  3. Chen GY, Nunez G (2010) Sterile inflammation: sensing and reacting to damage. Nat Rev Immunol 10:826–837. doi: 10.1038/nri2873 CrossRefPubMedPubMedCentralGoogle Scholar
  4. Couto Alves A et al (2013) Dysregulation of complement system and CD4+ T cell activation pathways implicated in allergic response. PLoS One 8:e74821. doi: 10.1371/journal.pone.0074821 CrossRefPubMedPubMedCentralGoogle Scholar
  5. Dinarello CA (2009) Immunological and inflammatory functions of the interleukin-1 family. Annu Rev Immunol 27:519–550. doi: 10.1146/annurev.immunol.021908.132612 CrossRefPubMedGoogle Scholar
  6. Dinarello CA (2011) Interleukin-1 in the pathogenesis and treatment of inflammatory diseases. Blood 117:3720–3732. doi: 10.1182/blood-2010-07-273417 CrossRefPubMedPubMedCentralGoogle Scholar
  7. Dostert C, Petrilli V, Van Bruggen R, Steele C, Mossman BT, Tschopp J (2008) Innate immune activation through Nalp3 inflammasome sensing of asbestos and silica. Science 320:674–677. doi: 10.1126/science.1156995 CrossRefPubMedPubMedCentralGoogle Scholar
  8. Doyle SL et al (2012) NLRP3 has a protective role in age-related macular degeneration through the induction of IL-18 by drusen components. Nat Med 18:791–798. doi: 10.1038/nm.2717 CrossRefPubMedPubMedCentralGoogle Scholar
  9. Duewell P et al (2010) NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals. Nature 464:1357–1361. doi: 10.1038/nature08938 CrossRefPubMedPubMedCentralGoogle Scholar
  10. Fang C, Zhang X, Miwa T, Song WC (2009) Complement promotes the development of inflammatory T-helper 17 cells through synergistic interaction with Toll-like receptor signaling and interleukin-6 production. Blood 114:1005–1015. doi: 10.1182/blood-2009-01-198283 CrossRefPubMedPubMedCentralGoogle Scholar
  11. Fann DY et al (2013) Intravenous immunoglobulin suppresses NLRP1 and NLRP3 inflammasome-mediated neuronal death in ischemic stroke. Cell Death Dis 4:e790. doi: 10.1038/cddis.2013.326 CrossRefPubMedGoogle Scholar
  12. Flower O, Smith M (2011) The acute management of intracerebral hemorrhage. Curr Opin Crit Care 17:106–114. doi: 10.1097/MCC.0b013e328342f823 CrossRefPubMedGoogle Scholar
  13. Fogal B, Li J, Lobner D, McCullough LD, Hewett SJ (2007) System x(c)- activity and astrocytes are necessary for interleukin-1 beta-mediated hypoxic neuronal injury. The Journal of neuroscience : the official journal of the Society for Neuroscience 27:10094–10105. doi: 10.1523/JNEUROSCI.2459-07.2007 CrossRefGoogle Scholar
  14. Forte A, Cipollaro M, Cascino A, Galderisi U (2005) Small interfering RNAs and antisense oligonucleotides for treatment of neurological diseases. Curr Drug Targets 6:21–29CrossRefPubMedGoogle Scholar
  15. Fountaine TM, Wood MJ, Wade-Martins R (2005) Delivering RNA interference to the mammalian brain. Current gene therapy 5:399–410CrossRefPubMedGoogle Scholar
  16. Fure H, Nielsen EW, Hack CE, Mollnes TE (1997) A neoepitope-based enzyme immunoassay for quantification of C1-inhibitor in complex with C1r and C1s. Scand J Immunol 46:553–557CrossRefPubMedGoogle Scholar
  17. Garrett MC et al (2009) Synergistic neuroprotective effects of C3a and C5a receptor blockade following intracerebral hemorrhage. Brain Res 1298:171–177. doi: 10.1016/j.brainres.2009.04.047 CrossRefPubMedGoogle Scholar
  18. Goldmann T, Tay TL, Prinz M (2013) Love and death: microglia, NLRP3 and the Alzheimer’s brain. Cell Res 23:595–596. doi: 10.1038/cr.2013.24 CrossRefPubMedPubMedCentralGoogle Scholar
  19. Goldstein JN, Gilson AJ (2011) Critical care management of acute intracerebral hemorrhage. Curr Treat Options Neurol 13:204–216. doi: 10.1007/s11940-010-0109-2 CrossRefPubMedPubMedCentralGoogle Scholar
  20. Halle A et al (2008) The NALP3 inflammasome is involved in the innate immune response to amyloid-beta. Nat Immunol 9:857–865. doi: 10.1038/ni.1636 CrossRefPubMedPubMedCentralGoogle Scholar
  21. Hanamsagar R, Hanke ML, Kielian T (2012) Toll-like receptor (TLR) and inflammasome actions in the central nervous system. Trends Immunol 33:333–342. doi: 10.1016/ CrossRefPubMedPubMedCentralGoogle Scholar
  22. Heeringa SF, Cohen CD (2012) Kidney diseases caused by complement dysregulation: acquired, inherited, and still more to come. Clinical & developmental immunology 2012:695131. doi: 10.1155/2012/695131 CrossRefGoogle Scholar
  23. Hoegen T et al (2011) The NLRP3 inflammasome contributes to brain injury in pneumococcal meningitis and is activated through ATP-dependent lysosomal cathepsin B release. J Immunol 187:5440–5451. doi: 10.4049/jimmunol.1100790 CrossRefPubMedGoogle Scholar
  24. Hwang BY et al (2011) Advances in neuroprotective strategies: potential therapies for intracerebral hemorrhage. Cerebrovasc Dis 31:211–222. doi: 10.1159/000321870 CrossRefPubMedGoogle Scholar
  25. Jiang Y, Ma J, Li H, Liu Y, You C (2015) Effect of apolipoprotein C3 genetic polymorphisms on serum lipid levels and the risk of intracerebral hemorrhage. Lipids Health Dis 14:48. doi: 10.1186/s12944-015-0047-9 CrossRefPubMedPubMedCentralGoogle Scholar
  26. Laudisi F et al (2013) Cutting edge: the NLRP3 inflammasome links complement-mediated inflammation and IL-1beta release. J Immunol 191:1006–1010. doi: 10.4049/jimmunol.1300489 CrossRefPubMedPubMedCentralGoogle Scholar
  27. Li G et al (2013) Neuroprotective effects of Argatroban and C5a receptor antagonist (PMX53) following intracerebral hemorrhage. Clin Exp Immunol. doi: 10.1111/cei.12220 Google Scholar
  28. Lin S et al (2012) Heme activates TLR4-mediated inflammatory injury via MyD88/TRIF signaling pathway in intracerebral hemorrhage. J Neuroinflammation 9:46. doi: 10.1186/1742-2094-9-46 PubMedPubMedCentralGoogle Scholar
  29. Lint TF, Behrends CL, Baker PJ, Gewurz H (1976) Activation of the complement attack mechanism in the fluid phase and its control by C567-INH: lysis of normal erythrocytes initiated by zymosan, endotoxin, and immune complexes. J Immunol 117:1440–1446PubMedGoogle Scholar
  30. Lok J et al (2012) Neuregulin-1 effects on endothelial and blood-brain-barrier permeability after experimental injury. Translational stroke research 3(Suppl 1):S119–S124. doi: 10.1007/s12975-012-0157-x CrossRefPubMedPubMedCentralGoogle Scholar
  31. Lu A, Tang Y, Ran R, Ardizzone TL, Wagner KR, Sharp FR (2006) Brain genomics of intracerebral hemorrhage. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism 26:230–252. doi: 10.1038/sj.jcbfm.9600183 CrossRefGoogle Scholar
  32. Mariathasan S, Monack DM (2007) Inflammasome adaptors and sensors: intracellular regulators of infection and inflammation. Nat Rev Immunol 7:31–40. doi: 10.1038/nri1997 CrossRefPubMedGoogle Scholar
  33. Martinon F, Petrilli V, Mayor A, Tardivel A, Tschopp J (2006) Gout-associated uric acid crystals activate the NALP3 inflammasome. Nature 440:237–241. doi: 10.1038/nature04516 CrossRefPubMedGoogle Scholar
  34. Masada T, Hua Y, Xi G, Yang GY, Hoff JT, Keep RF (2001) Attenuation of intracerebral hemorrhage and thrombin-induced brain edema by overexpression of interleukin-1 receptor antagonist. J Neurosurg 95:680–686. doi: 10.3171/jns.2001.95.4.0680 CrossRefPubMedGoogle Scholar
  35. Mollnes TE et al (2002) Essential role of the C5a receptor in E coli-induced oxidative burst and phagocytosis revealed by a novel lepirudin-based human whole blood model of inflammation. Blood 100:1869–1877PubMedGoogle Scholar
  36. Nakamura T, Xi G, Hua Y, Schallert T, Hoff JT, Keep RF (2004) Intracerebral hemorrhage in mice: model characterization and application for genetically modified mice. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism 24:487–494. doi: 10.1097/00004647-200405000-00002 CrossRefGoogle Scholar
  37. Petrilli V, Dostert C, Muruve DA, Tschopp J (2007) The inflammasome: a danger sensing complex triggering innate immunity. Curr Opin Immunol 19:615–622. doi: 10.1016/j.coi.2007.09.002 CrossRefPubMedGoogle Scholar
  38. Qureshi AI, Mendelow AD, Hanley DF (2009) Intracerebral haemorrhage. Lancet 373:1632–1644. doi: 10.1016/S0140-6736(09)60371-8 CrossRefPubMedPubMedCentralGoogle Scholar
  39. Rathinam VA, Vanaja SK, Fitzgerald KA (2012) Regulation of inflammasome signaling. Nat Immunol 13:333–342. doi: 10.1038/ni.2237 CrossRefPubMedPubMedCentralGoogle Scholar
  40. Ribo M, Grotta JC (2006) Latest advances in intracerebral hemorrhage. Current neurology and neuroscience reports 6:17–22CrossRefPubMedGoogle Scholar
  41. Rynkowski MA et al (2009) C3a receptor antagonist attenuates brain injury after intracerebral hemorrhage. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism 29:98–107. doi: 10.1038/jcbfm.2008.95 CrossRefGoogle Scholar
  42. Samstad EO et al (2014) Cholesterol crystals induce complement-dependent inflammasome activation and cytokine release. J Immunol 192:2837–2845. doi: 10.4049/jimmunol.1302484 CrossRefPubMedPubMedCentralGoogle Scholar
  43. Sansing LH, Kaznatcheeva EA, Perkins CJ, Komaroff E, Gutman FB, Newman GC (2003) Edema after intracerebral hemorrhage: correlations with coagulation parameters and treatment. J Neurosurg 98:985–992. doi: 10.3171/jns.2003.98.5.0985 CrossRefPubMedGoogle Scholar
  44. Savage CD, Lopez-Castejon G, Denes A, Brough D (2012) NLRP3-inflammasome activating DAMPs stimulate an inflammatory response in glia in the absence of priming which contributes to brain inflammation after injury. Front Immunol 3:288. doi: 10.3389/fimmu.2012.00288 CrossRefPubMedPubMedCentralGoogle Scholar
  45. Srinivasan D, Yen JH, Joseph DJ, Friedman W (2004) Cell type-specific interleukin-1beta signaling in the CNS. The Journal of neuroscience : the official journal of the Society for Neuroscience 24:6482–6488. doi: 10.1523/JNEUROSCI.5712-03.2004 CrossRefGoogle Scholar
  46. Stahel PF et al (2000) Intracerebral complement C5a receptor (CD88) expression is regulated by TNF and lymphotoxin-alpha following closed head injury in mice. J Neuroimmunol 109:164–172CrossRefPubMedGoogle Scholar
  47. Strowig T, Henao-Mejia J, Elinav E, Flavell R (2012) Inflammasomes in health and disease. Nature 481:278–286. doi: 10.1038/nature10759 CrossRefPubMedGoogle Scholar
  48. Stutz A, Golenbock DT, Latz E (2009) Inflammasomes: too big to miss. J Clin Invest 119:3502–3511. doi: 10.1172/JCI40599 CrossRefPubMedPubMedCentralGoogle Scholar
  49. Sukumari-Ramesh S, Alleyne CH Jr (2016) Post-injury administration of tert-butylhydroquinone attenuates acute neurological injury after intracerebral hemorrhage in mice. Journal of molecular neuroscience : MN 58:525–531. doi: 10.1007/s12031-016-0722-y CrossRefPubMedGoogle Scholar
  50. Suresh R, Chandrasekaran P, Sutterwala FS, Mosser DM (2016) Complement-mediated ‘bystander’ damage initiates host NLRP3 inflammasome activation. J Cell Sci 129:1928–1939. doi: 10.1242/jcs.179291 CrossRefPubMedGoogle Scholar
  51. Sutterwala FS, Haasken S, Cassel SL (2014) Mechanism of NLRP3 inflammasome activation. Ann N Y Acad Sci 1319:82–95. doi: 10.1111/nyas.12458 CrossRefPubMedPubMedCentralGoogle Scholar
  52. van Asch CJ, Luitse MJ, Rinkel GJ, van der Tweel I, Algra A, Klijn CJ (2010) Incidence, case fatality, and functional outcome of intracerebral haemorrhage over time, according to age, sex, and ethnic origin: a systematic review and meta-analysis. Lancet Neurol 9:167–176. doi: 10.1016/S1474-4422(09)70340-0 CrossRefPubMedGoogle Scholar
  53. Wei P, You C, Jin H, Chen H, Lin B (2014) Correlation between serum IL-1beta levels and cerebral edema extent in a hypertensive intracerebral hemorrhage rat model. Neurol Res 36:170–175. doi: 10.1179/1743132813Y.0000000292 CrossRefPubMedGoogle Scholar
  54. Wu B, Ma Q, Khatibi N, Chen W, Sozen T, Cheng O, Tang J (2010) Ac-YVAD-CMK decreases blood-brain barrier degradation by inhibiting caspase-1 activation of interleukin-1beta in intracerebral hemorrhage mouse model. Translational stroke research 1:57–64. doi: 10.1007/s12975-009-0002-z CrossRefPubMedPubMedCentralGoogle Scholar
  55. Xi G, Keep RF, Hoff JT (2002) Pathophysiology of brain edema formation. Neurosurg Clin N Am 13:371–383CrossRefPubMedGoogle Scholar
  56. Yang S et al (2006) The role of complement C3 in intracerebral hemorrhage-induced brain injury. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism 26:1490–1495. doi: 10.1038/sj.jcbfm.9600305 CrossRefGoogle Scholar
  57. Yang F et al (2014) NLRP3 deficiency ameliorates neurovascular damage in experimental ischemic stroke. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism. doi: 10.1038/jcbfm.2013.242 Google Scholar
  58. Yuen CM, Chiu CA, Chang LT, Liou CW, Lu CH, Youssef AA, Yip HK (2007) Level and value of interleukin-18 after acute ischemic stroke. Circulation journal : official journal of the Japanese Circulation Society 71:1691–1696CrossRefGoogle Scholar
  59. Zhang X et al (2007) Regulation of Toll-like receptor-mediated inflammatory response by complement in vivo. Blood 110:228–236. doi: 10.1182/blood-2006-12-063636 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Sheng-Tao Yao
    • 1
  • Fang Cao
    • 1
  • Jia-Lin Chen
    • 2
  • Wei Chen
    • 1
  • Rui-Ming Fan
    • 1
  • Gang Li
    • 1
  • You-Chao Zeng
    • 1
  • Song Jiao
    • 1
  • Xiang-Ping Xia
    • 1
  • Chong Han
    • 1
  • Qi-Shan Ran
    • 1
  1. 1.Department of Cerebrovascular DiseaseThe First Affiliated Hospital of Zunyi Medical CollegeZunyiChina
  2. 2.Department of NeonatalThe Third Affiliated Hospital of Zunyi Medical CollegeZunyiChina

Personalised recommendations