Role of mitochondria ROS generation in ethanol-induced NLRP3 inflammasome activation and cell death in astroglial cells

Abstract

Toll-like receptors (TLRs) and NOD-like receptors (NLRs) are innate immunity sensors that provide an early/effective response to pathogenic or injury conditions. We have reported that ethanol-induced TLR4 activation triggers signaling inflammatory responses in glial cells, causing neuroinflammation and brain damage. However, it is uncertain if ethanol is able to activate NLRs/inflammasome in astroglial cells, which is the mechanism of activation, and whether there is crosstalk between both immune sensors in glial cells. Here we show that chronic ethanol treatment increases the co-localization of caspase-1 with GFAP+ cells, and up-regulates IL-1β and IL-18 in the frontal medial cortex in WT, but not in TLR4 knockout mice. We further show that cultured cortical astrocytes expressed several inflammasomes (NLRP3, AIM2, NLRP1, and IPAF), although NLRP3 mRNA is the predominant form. Ethanol, as ATP and LPS treatments, up-regulates NLRP3 expression, and causes caspase-1 cleavage and the release of IL-1β and IL-18 in astrocytes supernatant. Ethanol-induced NLRP3/caspase-1 activation is mediated by mitochondrial (m) reactive oxygen species (ROS) generation because when using a specific mitochondria ROS scavenger, the mito-TEMPO (500 μM) or NLRP3 blocking peptide (4 μg/ml) or a specific caspase-1 inhibitor, Z-YVAD-FMK (10 μM), abrogates mROS release and reduces the up-regulation of IL-1β and IL-18 induced by ethanol or LPS or ATP. Confocal microscopy studies further confirm that ethanol, ATP or LPS promotes NLRP3/caspase-1 complex recruitment within the mitochondria to promote cell death by caspase-1-mediated pyroptosis, which accounts for ≈73% of total cell death (≈22%) and the remaining (≈25%) die by caspase-3-dependent apoptosis. Suppression of the TLR4 function abrogates most ethanol effects on NLRP3 activation and reduces cell death. These findings suggest that NLRP3 participates, in ethanol-induced neuroinflammation and highlight the NLRP3/TLR4 crosstalk in ethanol-induced brain injury.

FABP4/aP2 Regulates Macrophage Redox Signaling and Inflammasome Activation via Control of UCP2

“…It is also interesting that the most robust changes observed in the different cell lines consistently came from LonP1, which specifically recognizes oxidized proteins and targets them for degradation (35). Compromising homeostasis of the mitochondrion can lead to apoptosis, and one of the primary downstream effectors of the mtUPR in immune cells is activation on the inflammasome (22,38,41,42). This was prevented in cells lacking FABP4/aP2, as observed from the decrease in mRNA and protein levels of the inflammasome members, as well as its activation (IL-1␤ secretion).…”

“…The bioactive form of IL-1␤ must be generated by inflammasome-dependent cleavage and secretion, which can be triggered by ROS (as well as other sources) through the mitochondrial intrinsic apoptotic pathway (22,38). To assess activation of the inflammasome in the FABP4/aP2 system, IL-1␤ secretion was evaluated in response to either pharmacologic or genetic ablation of FABP4/aP2.…”

The Neuroimmune Transcriptome and Alcohol Dependence: Potential for Targeted Therapies

“…Some studies have identified the specific inflammatory mediators released in response to ethanol. For example, acute ethanol treatment of cultured astrocytes causes upregulation of iNOS and COX-2 [56], and chronic ethanol activates the NLRP3 inflammasome in astrocytes from cerebral cortex and in primary cultures of astrocytes [93]. Ethanol also promotes TLR4 signaling in astrocytes and microglia, which leads to upregulation of inflammatory cytokines, including IL-1β, TNF-α, IL-6, iNOS and COX-2 [63].…”

Microglial NLRP3 Activity in Alzheimer's Disease

“…However, other studies disagree with these findings and suggest NLRP3 activation in astrocytes after CNS injury. Therefore, further studies are needed to address these issues [43,44].…”