Mitochondria and NLRP3 Inflammasome Interplay: Comparison
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Mitochondria are the power plants of the cell responsible not only for the generation of cellular energy required for myriad functions but also are important hubs for metabolism, reactive oxygen species (ROS) generation, and Ca2+ homeostasis. Inflammasomes are a group of intracellular multicomplexes located in the cytosol which detect PAMPs and DAMPs and produce the activation, maturation, and release of pro-inflammatory cytokines (including IL-1β and IL-18).

  • NLRP3 inflammasome
  • mitochondria
  • steatohepatitis

1. Introduction

Alcoholic (ASH) and nonalcoholic steatohepatitis (NASH) are two of the most common causes of chronic liver disease worldwide. ASH is caused by chronic alcohol consumption, while NASH is associated with dietary habits and obesity and linked to insulin resistance and type 2 diabetes [1][2]. Although ASH and NASH differ in their etiology, both pathologies are characterized by the excess of fat deposition and lipotoxicity leading to inflammation, hepatocellular ballooning, and fibrogenesis [3][4]. Despite significant advances in the identification of players mediating the transition from steatosis to steatohepatitis, unfortunately the therapeutic options for ASH and NASH are limited, due to our incomplete understanding of the molecular mechanisms contributing to ASH and NASH development.
Mitochondria are the power plants of the cell responsible not only for the generation of cellular energy required for myriad functions but also are important hubs for metabolism, reactive oxygen species (ROS) generation, and Ca2+ homeostasis. Associated with the role of energy production, mitochondria consume large amounts of molecular oxygen in the respiratory chain and, hence, are critical players in the onset of oxidative stress, which is accentuated by the disturbance of mitochondrial function related to hepatocyte injury in ASH and NASH [5]. As oxidative stress reflects the imbalance between oxidants and antioxidants defense, the impairment in the levels and/or activity of mitochondrial antioxidant strategies, including disruption of the GSH redox cycle, enhances the accumulation of ROS and increases the susceptibility of the cells to inflammatory cytokines [6].
Furthermore, mitochondrial dysfunction can activate the nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome, leading to inflammation [7]. Inflammasomes are multiprotein immune complexes that are activated in response to pathogen-associated and danger-associated molecular patterns (PAMPs and DAMPs), such as lipopolysaccharide (LPS) and cholesterol crystals [8]. The activation of NLRP3 inflammasome followed by caspase-1 activation allows the release of proinflammatory cytokines, such as IL-1β and IL-18, into the extracellular space that promote inflammatory cell death (pyroptosis), leading to an increase in liver inflammation, steatosis, and fibrosis [9].

2. Overview of Mitochondria and NLRP3 Inflammasome Interplay

Inflammasomes are a group of intracellular multicomplexes located in the cytosol which detect PAMPs and DAMPs and produce the activation, maturation, and release of pro-inflammatory cytokines (including IL-1β and IL-18) [10][11]. Among the family of inflammasomes, the best characterized are NLRP1, NLRP3, NLRC4, and absent in melanoma 2 (AIM2) [12]. NLRP3, the best-studied member of the inflammasome family, is a multicomplex formed by NLRP3, adaptor apoptosis speck protein (ASC), and pro-caspase-1 [13]. Activation of the NLRP3 inflammasome can occur through two signal steps [8][14]. The priming signal is provided by microbial components (LPS) or endogenous cytokines (tumor necrosis factor alpha, TNF-α; IL-1β), followed by the binding to toll-like receptors (such as TLR4), leading to the activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), which in turn contributes to the transcription and translation of NLRP3. Then, NLRP3 undergoes post-translational modifications, resulting in its activation. The activation of the NLRP3 inflammasome leads to the subsequent activation of caspase 1, which can then cleave inactive pro-IL-1β and pro-IL-18 into active IL-1β and IL-18, respectively. The activation of caspase-1 also cleaves gasdermin D (GSDMD) into a N-terminal fragment, which transfers to the cell membrane for specific interactions with lipids. Cells undergo morphological changes, including plasma membrane rupture and pore formation, loss of ionic gradients, and osmosis resulting in cellular swelling due to water entry, and are finally lysed, causing the release of intracellular pro-inflammatory molecules (IL-1β and IL-18), which subsequently stimulate secondary cytokine production [15]. The signals for the inflammasome activation include extracellular stimuli such as adenosine triphosphate (ATP), pore-forming toxins, RNA viruses, cholesterol crystals, uric acid, and amyloid β [8][14]. There are three hypotheses regarding the activation of NLRP3 inflammasome: (a) the destabilization of the ion outflow (K+, Ca2+, and H+), creating holes in the cell membrane; (b) the release of ROS by damaged mitochondria; and (c) the rupture of lysosomes that produce the leak of the lysosomal enzyme cathepsin B [8]. Mitochondria are the main source of ROS (mtROS) (Figure 1).
Figure 1. Scheme of the pathways associated with NRLP3 inflammasome activation and mitochondrial dysfunction. PAMPs and DAMPs are recognized by TLR, as priming signal, and activate NF-κB signaling pathway, promoting the transcription of pro-IL-1β and NLRP3, while an activation signal initiates the formation of NLRP3/ASC/pro-caspase-1 complex. Subsequently, the active form of caspase-1 cleaves pro-IL-1β to mature IL-1β and GSDMD into a N-terminal fragment (N-GSDMD). N-GSDMD produces pores in the membrane, allowing the release of IL-1β into the extracellular space. The mechanisms involved in NLRP3 activation include: the destabilization of the ion outflow (K+ efflux/Ca2+ influx) creating holes in the cell membrane; the release of ROS by damaged mitochondria produced by excessive electron flow; an inhibited mitophagy and mtDNA oxidation; and the release of cathepsins by damaged lysosomes. The NLRP3 inflammasome complex activation is located in MAMs, where cardiolipin translocation and Ca2+ influx by VDAC occurs, and facilitates the assembly of the NLRP3 inflammasome. ASC, adaptor apoptosis speck protein; DAMPS, damage-associated molecular patterns; GSDMD, gasdermin D; IL-1β, interleukin-1β; MAM, mitochondria-associated membranes; NF-kB, nuclear factor kappa B; NLRP3, NLR family pyrin domain containing 3; PAMPs, pathogen-associated molecular patterns; ROS, reactive oxygen species; TLR, toll-like receptor.
Mitochondria are intracellular organelles with a double membrane that delimits the intermembrane space and the mitochondrial matrix. Both spaces have different and important functions for the cellular metabolism and homeostasis. In the soluble matrix, importantly, metabolic processes, such as the tricarboxylic acid cycle (TCA), fatty acid oxidation, and urea synthesis, take place [16]. Mitochondria are the main consumers of molecular oxygen in the cell in the respiratory chain, localized in the inner membrane and composed by the complexes I, II, III, and IV, cytochrome c, and coenzyme Q [16]. The mitochondrial respiratory chain is involved in the generation of ATP and in the homeostasis of ROS. Increased metabolic rates, hypoxia, or membrane damage are stress inducers of mitochondrial ROS production [17]. Excessive electron flow to the respiratory chain results in an enhanced generation of superoxide anion, hydroxyl radicals, and hydrogen peroxide. These free radicals and other oxidants are metabolized in mitochondria by the existence of efficient antioxidant strategy systems, comprised of several enzymes, such as superoxide dismutases (SODs), glutathione peroxidase (GSH-Px) and reductase (GSSG-Rx), and peroxiredoxins/thioredoxins, as well as non-enzymatic antioxidants (vitamin A/C/E and GSH) [18][19][20]. GSH is particularly crucial, as it is part—along with GSH-Px and GSH-Rx—of the GSH redox cycle that detoxifies hydrogen peroxide and other lipid peroxides. When the production of ROS exceeds the capacity of these antioxidant defenses, or if an antioxidant balance is not ensured, free radicals and ROS can accumulate, with the risk of damaging mitochondrial components, such as the mtDNA [21]. Oxidized mitochondrial DNA (ox-mtDNA) is released to the cytosol, where it can directly bind NLRP3 inflammasome, triggering its activation [22]. Furthermore, cardiolipin, a mitochondria-specific phospholipid, translocates from the inner to the outer mitochondrial membrane and produces the oligomerization and activation of the NLRP3 inflammasome in mitochondria-associated membranes (MAM) [23]. MAMs are specific sites where mitochondria and ER contact and are believed to play a critical role in the transfer of lipids and Ca2+ between ER and mitochondria [24]. Importantly, as mitochondria have been shown to co-localize with the NLRP3 [7], mitochondria dysfunction stands as an important factor that facilitates the assembly of the NLRP3 inflammasome [25]. In line with the link between mitochondria and NLRP3, Zhou et al. showed that the inhibition of complex I or III of the mitochondrial respiratory chain causes unprompted NLRP3 inflammasome activation [7]. Additional findings showed that the inhibition of mitophagy/autophagy results in the prolonged generation of ROS from damaged mitochondria, a process in which the sequestosome-1 (SQSTM1, also called p62) plays an essential role in ensuring mitochondrial quality control via mitophagy [26]. Besides the quantitative generation of ROS by mitochondria in the respiratory chain, there are other sources of ROS generation, including the β-oxidation pathways of mitochondria and peroxisomes, as well as the cytochrome P450 2E1 (CYP2E1) in the ER [27]. The link between the extramitochondrial generation of ROS and NLRP3 activation remains to be further established. Nevertheless, the activation of NLRP3 inflammasome has been implicated as a crucial factor in the development of different pathologies, such as metabolic syndrome, atherosclerosis, and neurodegenerative diseases, as well as inflammatory diseases, including ASH and NASH [28]. However, the exact link between mitochondrial dysfunction and its relationship with NLRP3 is not completely understood and requires further investigation (Figure 2).
Figure 2. Mechanism of crosstalk between mitochondria and inflammasome in ASH and NASH. Ethanol consumption and FFA- and cholesterol-enriched diets can lead to damaged intestinal barrier where PAMPs and DAMPs such as FFA or microbial products go through the disrupted tight junctions and promote NLRP3 inflammasome activation in liver cells. This triggers the activation of caspase-1, which mediates the cleavage of pro-IL-1β and GSDMD into their mature forms, which in turn promote hepatocellular death and the attraction and activation of KCs and HSCs, leading to inflammation and fibrosis. The metabolism of ethanol and FFA in hepatocytes induces an increment in ROS in the mitochondria that in turn triggers inflammasome activation. ADH, alcohol dehydrogenase; ALDH2, acetaldehyde dehydrogenase 2; ASC, adaptor apoptosis speck protein; DAMPS, damage-associated molecular patterns; FFA, free fatty acid; GSDMD, gasdermin D; HSC, hepatic stellate cells; IL-1β, interleukin-1β; KC, Kupffer cells; NADH/NAD+, oxidized and reduced nicotinamide adenine dinucleotide ratio; NLRP3, NLR family pyrin domain containing 3; NK, natural killer cells; PAMPs, pathogen-associated molecular patterns; ROS, reactive oxygen species; TCA, tricarboxylic acid cycle.

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