Abstract
Multi-protein complexes called inflammasomes have recently been identified and shown to contribute to cell death in tissue injury. Intravenous immunoglobulin (IVIg) is an FDA-approved therapeutic modality used for various inflammatory diseases. The objective of this study is to investigate dynamic responses of the NLRP1 and NLRP3 inflammasomes in stroke and to determine whether the NLRP1 and NLRP3 inflammasomes can be targeted with IVIg for therapeutic intervention. Primary cortical neurons were subjected to glucose deprivation (GD), oxygen–glucose deprivation (OGD) or simulated ischemia-reperfusion (I/R). Ischemic stroke was induced in C57BL/6J mice by middle cerebral artery occlusion, followed by reperfusion. Neurological assessment was performed, brain tissue damage was quantified, and NLRP1 and NLRP3 inflammasome protein levels were evaluated. NLRP1 and NLRP3 inflammasome components were also analyzed in postmortem brain tissue samples from stroke patients. Ischemia-like conditions increased the levels of NLRP1 and NLRP3 inflammasome proteins, and IL-1β and IL-18, in primary cortical neurons. Similarly, levels of NLRP1 and NLRP3 inflammasome proteins, IL-1β and IL-18 were elevated in ipsilateral brain tissues of cerebral I/R mice and stroke patients. Caspase-1 inhibitor treatment protected cultured cortical neurons and brain cells in vivo in experimental stroke models. IVIg treatment protected neurons in experimental stroke models by a mechanism involving suppression of NLRP1 and NLRP3 inflammasome activity. Our findings provide evidence that the NLRP1 and NLRP3 inflammasomes have a major role in neuronal cell death and behavioral deficits in stroke. We also identified NLRP1 and NLRP3 inflammasome inhibition as a novel mechanism by which IVIg can protect brain cells against ischemic damage, suggesting a potential clinical benefit of therapeutic interventions that target inflammasome assembly and activity.
Highlights
Cell Death and Disease conditions activate the inflammasome in neurons, we evaluated the temporal expression of all NLRP1 and NLRP3 inflammasome components in neurons subjected to simulated ischemia-reperfusion (I/R)
The levels of all major inflammasome components and effectors were increased in primary cortical neurons in response to glucose deprivation (GD), oxygen–glucose deprivation (OGD) and simulated I/R conditions including NLRP1, NLRP3, ASC, XIAP, and precursor caspases 1 and 11 (Figures 1a–f; Supplementary Figures 1–3)
Activation and oligomerization of the NLRP1 and NLRP3 receptors individually induces the formation of the NLRP1 and NLRP3 inflammasome, respectively, which activates both precursor caspase-1 and -11 into biologically active cleaved caspase-1 and 11.10 Following activation, caspase-1 cleaves both precursors IL-1b and IL-18 into biologically active mature pro-inflammatory cytokines, which are released into the extracellular environment.[25]
Summary
The NLRP1 and NLRP3 inflammasomes are cytosolic macromolecular complexes composed of the NLRP1/3 receptor, ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain), precursor caspase-1, precursor caspase-11 (homologous to precursor caspase-4 or 5 in humans) and/or XIAP (X-linked inhibitor of apoptosis).[8,9,10] Activation and homo-oligomerization of NLRP1 and NLRP3 receptors induces formation of the NLRP1 and NLRP3 inflammasomes, respectively, which convert precursor caspase-1 into cleaved caspase-1 via proximity-induced autoactivation.[6,10] Cleaved caspase-1 converts precursors of both IL-1b and IL-18 into biologically active mature pro-inflammatory cytokines that are released into the extracellular environment.[7,11,12] increased cleaved caspase-1 can initiate cell death directly via pyroptosis, or indirectly via apoptosis.[13,14] In stroke-related studies, reduced brain expression of mature IL-1b and IL-18 was shown in mice following cerebral ischemia, using an anti-NLRP1 antibody.[7] in caspase-1 knockout mice there was a reduction in mature IL-1b and IL-18 levels in association with a smaller infarct.[15] administration of an IL-1b-neutralizing antibody or IL-1 receptor antagonist reduced subarachnoid hemorrhagic injury.[16] the specific pathophysiological role of the NLRP1 and NLRP3 inflammasome in neuronal cell death following ischemic stroke remains to be established.
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