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

Abstract

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.

Selective NLRP3 (Pyrin Domain–Containing Protein 3) Inflammasome Inhibitor Reduces Brain Injury After Intracerebral Hemorrhage

“…Mechanistic studies have shown an essential role for the NLRP3 inflammasome in brain injury and neuroinflammation after ICH. 9 – 11 The activation of NLRP3 inflammasome facilitates caspase-1 activation and IL-1β processing, leading to the amplification of the inflammatory response, which culminates in the expansion of perihematomal edema, and thereby exacerbating hemorrhagic brain injury. 9 – 11 As a small molecule, the NLRP3 inflammasome inhibitor, MCC950, was recently found to selectively inhibit NLRP3 inflammasome formation and reduce pyroptosis and IL-1β signaling.…”

“… 9 – 11 The activation of NLRP3 inflammasome facilitates caspase-1 activation and IL-1β processing, leading to the amplification of the inflammatory response, which culminates in the expansion of perihematomal edema, and thereby exacerbating hemorrhagic brain injury. 9 – 11 As a small molecule, the NLRP3 inflammasome inhibitor, MCC950, was recently found to selectively inhibit NLRP3 inflammasome formation and reduce pyroptosis and IL-1β signaling. 17 In the present study, our results confirmed the ability of MCC950 to effectively inhibit the activation of NLRP3 inflammasome components and IL-1β production in experimental ICH.…”

“…Recent evidence indicates that the ICH-induced activation of NLRP3 inflammasome can amplify the inflammatory response by releasing IL-1β and promoting neutrophil infiltration, thereby exacerbating brain edema. 9 – 11 Although previous studies provide mechanistic insights into NLRP3 as a promising target for restricting neuroinflammation and brain edema after ICH, they do not reflect clinical application because they involve either knockout models or use of pharmacological agents with limited potency and nonspecificity preceding ICH induction. 9 – 11 For clinical translation of prior experimental outcomes, preclinical testing in relevant models is mandatory.…”

“… 9 – 11 Although previous studies provide mechanistic insights into NLRP3 as a promising target for restricting neuroinflammation and brain edema after ICH, they do not reflect clinical application because they involve either knockout models or use of pharmacological agents with limited potency and nonspecificity preceding ICH induction. 9 – 11 For clinical translation of prior experimental outcomes, preclinical testing in relevant models is mandatory. However, this option has been precluded by the lack of selective, potent NLRP3 inflammasome inhibitors.…”

Intracellular complement activation—An alarm raising mechanism?

“…Likewise, in T-cells, a gene set enrichment analysis revealed a number of inflammasome-related genes to be upregulated following TCR/CD46 co-stimulation, including IL1-beta and NLRP3 [53]. In mice complement was a crucial inducer of Il-1β expression, and C3 −/− knock-out mice display a reduced inflammasome activation in microglial cells following brain inflammation [169].…”