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.
This is a preview of subscription content, log in to check access.
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.
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.
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.2459-07.2007CrossRefGoogle Scholar
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
Fountaine TM, Wood MJ, Wade-Martins R (2005) Delivering RNA interference to the mammalian brain. Current gene therapy 5:399–410CrossRefPubMedGoogle Scholar
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
Heeringa SF, Cohen CD (2012) Kidney diseases caused by complement dysregulation: acquired, inherited, and still more to come. Clinical & developmental immunology 2012:695131. doi:10.1155/2012/695131CrossRefGoogle Scholar
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
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.9600183CrossRefGoogle Scholar
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
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:10.1097/00004647-200405000-00002CrossRefGoogle Scholar
Ribo M, Grotta JC (2006) Latest advances in intracerebral hemorrhage. Current neurology and neuroscience reports 6:17–22CrossRefPubMedGoogle Scholar
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.2008.95CrossRefGoogle Scholar
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.5712-03.2004CrossRefGoogle Scholar
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
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)70340-0CrossRefPubMedGoogle Scholar
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.9600305CrossRefGoogle Scholar
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.2013.242Google Scholar
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