NASH is an Inflammatory Disorder: Pathogenic, Prognostic and Therapeutic Implications

Abstract

While non-alcoholic fatty liver disease (NAFLD) is highly prevalent (15% to 45%) in modern societies, only 10% to 25% of cases develop hepatic fibrosis leading to cirrhosis, end-stage liver disease or hepatocellular carcinoma. Apart from pre-existing fibrosis, the strongest predictor of fibrotic progression in NAFLD is steatohepatitis or non-alcoholic steatohepatitis (NASH). The critical features other than steatosis are hepatocellular degeneration (ballooning, Mallory hyaline) and mixed inflammatory cell infiltration. While much is understood about the relationship of steatosis to metabolic factors (over-nutrition, insulin resistance, hyperglycemia, metabolic syndrome, hypoadiponectinemia), less is known about inflammatory recruitment, despite its importance for the perpetuation of liver injury and fibrogenesis. In this review, we present evidence that liver inflammation has prognostic significance in NAFLD. We then consider the origins and components of liver inflammation in NASH. Hepatocytes injured by toxic lipid molecules (lipotoxicity) play a central role in the recruitment of innate immunity involving Toll-like receptors (TLRs), Kupffer cells (KCs), lymphocytes and neutrophils and possibly inflammasome. The key pro-inflammatory signaling pathways in NASH are nuclear factor-kappa B (NF-κB) and c-Jun N-terminal kinase (JNK). The downstream effectors include adhesion molecules, chemokines, cytokines and the activation of cell death pathways leading to apoptosis. The upstream activators of NF-κB and JNK are more contentious and may depend on the experimental model used. TLRs are strong contenders. It remains possible that inflammation in NASH originates outside the liver and in the gut microbiota that prime KC/TLR responses, inflamed adipose tissue and circulating inflammatory cells. We briefly review these mechanistic considerations and project their implications for the effective treatment of NASH.

Keywords: Hepatic fibrosis; Non-alcoholic fatty liver disease; Non-alcoholic steatohepatitis.

Fig. 1

Excess lipid accumulation activates inflammatory pathways and induces insulin resistance. Extracellular free fatty acids (FFA) activate toll-like receptors (TLR), causing downstream activation of c-Jun N-terminal kinase (JNK) and IκB kinase (IKK) complex (composed of IKKα, IKKβ and NF-κB essential modulator [NEMO]). IKK heterotrimeric holocomplex catalyzes downstream activation of nuclear factor-kappa B (NF-κB), allowing p65 (also known as RELA), a proinflammatory transcription factor, to enter the nucleus where it induces transcriptional expression of multiple proinflammatory chemokines (e.g., macrophage chemotactic protein 1 [MCP-1]), cytokines, and adhesion molecules (e.g., vascular cell adhesion molecule-1). Once activated, JNK activates c-Jun which is involved with hepatocellular cell death, and via formation of heterodimeric c-Jun:c-Fos forms the pro-inflammatory transcription factor, activator protein 1 (AP-1). In addition to TLR activation, some intracellular lipid molecules (Table 2) may result in JNK/NF-κB activation by formation of reactive oxygen species (ROS); ROS may arise from excessive β-oxidation of FFA, uncoupling of oxidative phosphorylation and mitochondrial damage caused by free cholesterol (FC) accumulation and crystallization. Alternatively, some intracellular lipids may induce endoplasmic reticulum (ER) stress, leading to JNK/NF-κB p65 activation (see Fig. 3 for more details). JNK activation can also phosphorylate insulin receptor substrates (IRS)-1 and -2, which by blocking insulin receptor signal transduction leads to insulin resistance. TNF-α, tumor necrosis factor-α; IL-1β, interleukin-1β.

Fig. 2

Inflammatory cell recruitment and localization around lipid-laden hepatocytes in HF-fed foz/foz mice with non-alcoholic steatohepatitis (NASH). (A) H&E-stained liver section from HF-fed (0.2% cholesterol) foz/foz mouse with NASH, showing several enlarged hepatocytes with macrosteatotic vacuoles, and at least one ballooned hepatocyte (bottom right). (B) Macrophages (F4/80 positive), and (C) neutrophils (myeloperoxidase positive) accumulate around hepatocytes showing macrosteatotic vacuoles. These livers contain large amounts of free cholesterol. Scale bars represent 20 µm.

Fig. 3

Mammalian unfolded protein response (UPR) pathways. The UPR is triggered by several events, including protein unfolding/misfolding, hypoxia, low adenosine triphosphate levels, ER calcium depletion, and protein/sterol over-expression, causing dissociation of 78 kDa glucose-regulated protein (GRP78) from the three UPR sensors, (A) inositol-requiring enzyme 1α (IRE1α), (B) protein kinase RNA-like endoplasmic reticulum kinase (PERK), and (C) activating transcription factor-6 (ATF6). Activated IRE1α undergoes dimerization and autophosphorylation to generate endogenous RNase activity; in turn, this is responsible for splice truncation of X-box binding protein 1 (XBP1S) mRNA. Additionally, IRE1α may also activate the extrinsic apoptosis pathway, in which tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2)-dependent downstream activation of c-Jun N-terminal kinase (JNK) and caspase-12 takes place. Once activated, PERK undergoes homodimerisation and autophosphorylation to activate eukaryotic translation initiation factor 2 (eIF2α). In turn, this induces ATF4 expression. Separately, dissociation of GRP78, allows ATF6 processing by the Golgi complex, where proteases S1P and S2P cleave an active 50 kDa (p50) ATF6 domain that is free to translocate to the nucleus. Xbp1s, ATF4 and ATF6, as well as other unlisted factors, are responsible for three dominant cell responses to UPR. The folding pathway induces increased expression of molecular chaperones, including GRP78, assisting in compensatory ER protein folding. Alternatively, the cell may respond by increasing ER-associated protein degradation (ERAD) pathway, whereby gene products target and degrade unfolded proteins in the ER. Prolonged UPR results in the activation of the intrinsic apoptosis pathway; this ATF6 and ATF4-dependent process induces C/EBP-homologous protein (CHOP) expression. In turn, CHOP inhibits B-cell lymphoma 2 and induces apoptosis.

Fig. 4

Mitophagy inhibits pathways of mitochondrial dysfunction and associated cell death and inflammation. Mitophagy restitutes physiological cell functioning by inhibiting mitochondrial-related cell death and/or injury arising either from the generation of reactive oxygen-species (ROS) or pro-inflammatory signals, or as a result of mitochondrial membrane permeability transition (MPT). During activation of the intrinsic apoptosis pathway, BH3-only protein members, including BAK and BAX, effect mitochondrial membrane permeabilisation (MOMP) and release of intermembrane space proteins, including cytochrome c which induces a downstream caspase cascade activation that leads to apoptosis. Alternatively, necrotic cell death may be initiated by cyclophilin D-dependent initiation of MPT pore. Once opened, MPT destroys the mitochondrial transmembrane potential (Δψm), thereby abrogating oxidative phosphorylation and exacerbating ROS generation. Excessive ROS formation can activate the NACHT, LRR and PYD domains-containing protein 3 (NALP3) inflammasome. Uncoupling of oxidative phosphorylation can also trigger MPT, during which mtDNA may undergo cytoplasmic translocation, leading to nuclear factor-kappa B (NF-κB) and interferon regulatory factor-dependent inflammatory pathway activation. Importantly, excess intra-mitochondrial ROS is able to mutate mitochondrial DNA (mtDNA), leading to premature aging and mitochondrial inefficiency post-replication (this in turn exacerbates ROS generation through impaired oxidative phosphorylation).

Fig. 5

Toll-like receptor (TLR) signalling involves JNK and NF-κB p65 activation. Toll-like receptors (TLR) constitute a family of receptors involved in pro-inflammatory signalling in the innate immune system, responsible for the recognition of pathogen-associated molecular patterns (PAMPs) and exogenous stimuli, such as pathogens, or endogenous agonists, such as sterile tissue damage; the later are termed danger-associated molecular patterns (DAMPs). Of the 9 known TLR receptors, four (TLR-3, -7, -8, and -9) are expressed on the endosomal membrane and are responsible for viral particle surveillance, including detection of deoxy-cytidylate-phosphate-deoxy-guanylate DNA (CpG-DNA), and single- and double-stranded RNA. The remaining TLRs are expressed on the plasma membrane and are responsible for the detection of extracellular microbial pathogens. Relevant PAMPs include: LPS, diacyl- and triacyl lipopeptides, and flagellin, as well as several DAMPs, including HMGB1. Activated TLR3, as well as TLR4, signal through adaptor protein TIR-domain-containing adapter-inducing interferon-β (TRIF), which in turn recruits RIP1 to activate the IKK complex, thereby activating nuclear factor-kappa B (NF-κB). The other TLRs signal through toll/interleukin-1 receptor domain containing adaptor protein (TIRAP) and myeloid differentiation factor 88 (Myd88). Activated Myd88 induces the recruitment of IL-1R-associated kinase (IRAK) 4, as well as IRAK1, which bind TRAF-6 and transforming growth factor-β activated kinase (TAK)-1. IRF5 and IRF7 are then recruited to the post-Myd88 protein complex. Interferon-regulatory factor 7 (IRF7) recruitment is dependent upon on TLR7 and TLR9 signalling. The IRAK1/4/TRAF6/TAK1/IRF5/7 complex is responsible for downstream Myd88-dependent activation of c-Jun N-terminal kinase (JNK) and NF-κB. TRAF, tumor necrosis factor (TNF) receptor-associated factor; MEKK, MAP kinase kinase kinase; MKK, mitogen-activated protein kinase kinase; ASK, apoptosis signal-regulating kinase.