Inflammasomes in chronic liver disease: Hepatic injury, fibrosis progression and systemic inflammation

Abstract

Chronic liver disease leads to hepatocellular injury that triggers a pro-inflammatory state in several parenchymal and non-parenchymal hepatic cell types, ultimately resulting in liver fibrosis, cirrhosis, portal hypertension and liver failure. Thus, an improved understanding of inflammasomes - as key molecular drivers of liver injury - may result in the development of novel diagnostic or prognostic biomarkers and effective therapeutics. In liver disease, innate immune cells respond to hepatic insults by activating cell-intrinsic inflammasomes via toll-like receptors and NF-κB, and by releasing pro-inflammatory cytokines (such as IL-1β, IL-18, TNF-α and IL-6). Subsequently, cells of the adaptive immune system are recruited to fuel hepatic inflammation and hepatic parenchymal cells may undergo gasdermin D-mediated programmed cell death, termed pyroptosis. With liver disease progression, there is a shift towards a type 2 inflammatory response, which promotes tissue repair but also fibrogenesis. Inflammasome activation may also occur at extrahepatic sites, such as the white adipose tissue in MASH (metabolic dysfunction-associated steatohepatitis). In end-stage liver disease, flares of inflammation (e.g., in severe alcohol-related hepatitis) that spark on a dysfunctional immune system, contribute to inflammasome-mediated liver injury and potentially result in organ dysfunction/failure, as seen in ACLF (acute-on-chronic liver failure). This review provides an overview of current concepts regarding inflammasome activation in liver disease progression, with a focus on related biomarkers and therapeutic approaches that are being developed for patients with liver disease.

Keywords: Kupffer cell; alcohol-related liver disease; cirrhosis; hepatic stellate cell; inflammasome; liver fibrosis; macrophage; metabolic dysfunction-associated steatotic liver disease; toll-like receptor, nod-like receptor.

Fig. 1.. Mechanism of inflammasome assembly and activation.

(A) NLRP3 inflammasome. PAMPs bind to the membrane TLR leading to the “priming” of the NLRP3 inflammasome by inducing de-ubiquitination in a non-transcriptional way^, and activating NF-κB signalling. The ubiquitination and phosphorylation of ASC completes the assembly of the NLRP3 inflammasome. The second step required for “activation” involves a second hit by a wide range of stimuli, such as extracellular ATP, urates, β-amyloid protein, and ROS, that subsequently result in intracellular “stress” events, such as excessive production of ROS, mitochondrial dysfunction, release of oxidized DNA, lysosomal dysfunction and cathepsin release, changes in intracellular calcium level, the formation of cell membrane pores, or potassium efflux. The mechanisms of NLRP3 activation involve the PYD domain of the NLRP3 protein which recruits the adaptor protein ASC, which then serves as a hinge for connecting the NLRP3 inflammasome complex to pro-CASP1 via the CARD. The assemble of NLRP3-ASC-proCASP1 complexes facilitates proximity-induced auto-processing and results in the formation of the active CASP1 enzyme, that produces IL-1β, IL-18 and GSDMD. The functional N-terminal GSDMD fragment binds to the cell membrane, oligomerises and creates a pore in the cell membrane causing ion gradient perturbations but also allowing for the release of selected molecules including mature IL-1β and IL-18.^– (B) NLRC4 inflammasome. Several bacterial components such as needle protein, rod or flagellin are sensed by the cell membrane complex T3SS. The ligand specificity is conferred by a range of receptors known as NAIPs, that connect the sensing signal of T3SS to the activation of NLRC4 inflammasome. From the seven genes that encode NAIPs, four are found in mice (NAIP1, NAIP2, NAIP5 and NAIP6) and only one in humans (hNAIP). The hNAIP senses all components of the T3SS, while each murine NAIP specifically binds to its cognate ligand.^– NLRC4 can directly recruit CASP1 through its CARD domain, so it may function in the absence of the ASC fragment. (C) AIM2 inflammasome. The AIM2 inflammasome is directly activated by bacterial or host defective double-stranded DNA (aging, activation of oncogenes, etc.). AIM2 lacks the CARD domain, so it requires ASC to recruit pro-caspase-1. The complex then oligomerises to form the AIM2 inflammasome, which can activate CASP1; (D) Non-canonical inflammasomes. The non-canonical activation of the inflammasome involves the presence of cytosolic LPS that recruits pro-CASP11 in mice and pro-CASP4 and pro-CASP5 in humans. The complex oligomerises and activates CASP11 (in mice) and CASP4/5 (in humans). The activated non-canonical caspases cannot directly process IL-1β and IL-18 but do so via alternative activation of the CASP1 enzyme. At the same time, the non-canonical caspases cleave GSDMD to its N-terminal active form, which leads to the formation of cell membrane pores and to a type of apoptotic cell death termed pyroptosis.^, ASC, apoptosis-associated speck-like protein containing a caspase recruitment domain; CARD, caspase recruitment domain; CASP, caspase; GSDMD, gasdermin D; IL-, interleukin-; LPS, lipopolysaccharide; NLRP3, Nod-like receptor pyrin domain containing-3; NAIPs, NLR family of apoptosis inhibitory proteins; PAMPs, pathogen-associated molecular patterns; ROS, reactive oxygen species; T3SS, type III secretion system; TLR, toll-like receptor.

Fig. 2.. Crosstalk between resident liver cells and innate immune cells in ACLD.

(1) In the early stages of ACLD, the central inflammatory stimuli (ROS, ATP, etc.) arise from damaged hepatocytes exposed to a causative (aetiological) factor such as ethanol, oxidized lipids, hepatitis viruses, etc.^, (2) Injured/damaged hepatocytes subsequently release molecules collectively known as DAMPs that can stimulate KCs and activate inflammasomes via the P2RX7 receptor (which senses extracellular ATP). (3) During late stages of ACLD, pathogens (i.e. LPS). leak through the damaged intestinal barrier into the portal venous circulation and finally reach the liver, (4) where they induce NLRP3 inflammasome priming via TLR4. (5) Release of pro-inflammatory cytokines through oligomerised gasdermin D pores in the cell membrane, first into the extracellular space and then into the liver sinusoid, determine (6) the recruitment of NK cells and BMDMs.^, The release of IFN-γ from NK cells leads to differentiation of BMDMs into type I macrophages, which further (7) activate quiescent hepatic stellate cells via the CXCL2/CXCR2 pathway, resulting in collagen production and extracellular matrix deposition and ultimately liver fibrosis. (8) Persistent inflammation of adipose tissue, characteristic of metabolic syndrome and MASH, is partially mediated by inflammasome activation in the infiltrative type I macrophages and interactions with CD4+ T lymphocytes. (9) The NLRP6 inflammasome displays a predominantly anti-inflammatory function, by maintaining the functionality of intestinal epithelium during alcohol-related injury. (10) Several therapeutic molecules have been developed that target components of the inflammasome pathway. ACLD, advanced chronic liver disease; BMDMs, bone marrow-derived macrophages; CXCL2, C-X-C motif chemokine ligand 2; CXCR2, CXCL2 receptor; DAMPs, damage-associated molecular patterns; HSC, hepatic stellate cell; IFN, interferon; KCs, Kupffer cells; LPS, lipopolysaccharide; MASH, metabolic dysfunction-associated steatohepatitis; NK, natural killer; NLRP3/6, Nod-like receptor pyrin domain containing-3/6; P2RX7, purinergic receptor 2X7; ROS, reactive oxygen species; TLR4, toll-like receptor 4.