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.
Highlights
Inflammation in the central nervous system (CNS), or neuroinflammation, is a key component of many neurological and neurodegenerative disorders characterized by lymphocyte/macrophage infiltration, glial activation, enhanced cytokine/chemokine production, demyelination and axonal loss (Sospedra and Martin, 2005; Pittock and Lucchinetti, 2007)
We have demonstrated that ethanol is capable of activating TLR4/IL-1RI receptors in astroglial and microglial cells to trigger TLR4 signaling and to produce cytokines induction (IL-1β, TNF-α, IL-6) and inflammatory mediators, which can lead to neuroinflammation and brain injury
If we consider that ASC is an adaptor protein required for the activation of both NLRP3 inflammasome and caspase-1 (Fernandes-Alnemri et al, 2007), the results indicate that by inducing NLRP3 inflammasome complex activation and ASC-pyroptosome formation, ethanol was capable of inducing the innate immune response
Summary
Inflammation in the central nervous system (CNS), or neuroinflammation, is a key component of many neurological and neurodegenerative disorders characterized by lymphocyte/macrophage infiltration, glial activation, enhanced cytokine/chemokine production, demyelination and axonal loss (Sospedra and Martin, 2005; Pittock and Lucchinetti, 2007). Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) are two major forms of innate immune sensors which provide immediate responses against pathogenic invasion, tissue injury and stress conditions. Both the TLRs and NLRs families of receptors are activated through the recognition of both conserved microbial structures, called pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). Unlike membrane-bound TLRs that sense PAMPs or DAMPs on the cell surface or within endosomes, NLRs recognize microbial molecules or DAMPs in the host cytosol Activation of these receptors induces the recruitment of innate immune cells, which initiates tissue repair processing and adaptive immune activation. Abnormalities in any of these innate sensor-mediated processes may cause excessive inflammation due to either hyper-responsive innate immune signaling or sustained compensatory adaptive immune activation
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