Cellular heterogeneity and plasticity during NAFLD progression

Abstract

Nonalcoholic fatty liver disease (NAFLD) is a progressive liver disease that can progress to nonalcoholic steatohepatitis (NASH), NASH-related cirrhosis, and hepatocellular carcinoma (HCC). NAFLD ranges from simple steatosis (or nonalcoholic fatty liver [NAFL]) to NASH as a progressive form of NAFL, which is characterized by steatosis, lobular inflammation, and hepatocellular ballooning with or without fibrosis. Because of the complex pathophysiological mechanism and the heterogeneity of NAFLD, including its wide spectrum of clinical and histological characteristics, no specific therapeutic drugs have been approved for NAFLD. The heterogeneity of NAFLD is closely associated with cellular plasticity, which describes the ability of cells to acquire new identities or change their phenotypes in response to environmental stimuli. The liver consists of parenchymal cells including hepatocytes and cholangiocytes and nonparenchymal cells including Kupffer cells, hepatic stellate cells, and endothelial cells, all of which have specialized functions. This heterogeneous cell population has cellular plasticity to adapt to environmental changes. During NAFLD progression, these cells can exert diverse and complex responses at multiple levels following exposure to a variety of stimuli, including fatty acids, inflammation, and oxidative stress. Therefore, this review provides insights into NAFLD heterogeneity by addressing the cellular plasticity and metabolic adaptation of hepatocytes, cholangiocytes, hepatic stellate cells, and Kupffer cells during NAFLD progression.

Keywords: Cholangiocytes; Kupffer cells; NAFLD; cellular plasticity; hepatic stellate cells; hepatocytes; heterogeneity; metabolic adaptation.

FIGURE 1

Metabolic changes and adaptive cellular plasticity induced by multiple factors during NAFLD progression. NAFLD is a complex disease influenced by a number of factors. The ‘first hit’ of NAFLD is excessive liver fat accumulation induced by insulin resistance. Hepatic lipid accumulation is closely associated with ^① increased hepatic FA uptake, ^② increased hepatic de novo FA synthesis, ^③ increased lipogenesis, ^④ decreased lipolysis, ^⑤ decreased FA β-oxidation, and ^⑥ decreased VLDL-TG secretion. The “second hit” of NAFLD is inflammation and oxidative stress. As well, NAFLD is developed and progressed by complex factors, such as endotoxins and other environmental factors. The liver consists of parenchymal hepatocytes and nonparenchymal cells (Kupffer cells, hepatic stellate cells, and endothelial cells), all of which have specialized functions. Transformation of NAFL to NASH is accelerated through cellular plasticity and adaptive metabolic changes regulated by the specific responses and crosstalk of these cells to various stimuli such as excessive intrahepatic FFAs, inflammation, and oxidative stress. Abbreviations: aHSCs, activated hepatic stellate cells; ApoB, apolipoprotein B; DAMPs, damage-associated molecular patterns; EMT, epithelial-to-mesenchymal transition; FFAs, free fatty acids; GLUT2, glucose transporter 2; LPS, lipopolysaccharide; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; qHSC, quiescent hepatic stellate cell; ROS, reactive oxygen species; TG, triglyceride; TLR4, Toll-like receptor 4.

FIGURE 2

Functionally different hepatocyte populations according to their location in the liver. Liver cells are mainly composed of hepatocytes, hepatic stellate cells (HSCs), Kupffer cells, and liver sinusoidal endothelial cells (LSECs). Additionally, there are cholangiocytes in bile ducts and smooth muscle cells near the central vein. Liver cells have specialized functions depending on their location although hepatocytes are morphologically similar. Namely, hepatocytes located in the portal vein and central vein have different functions, suggesting that hepatocytes in different zones exert different functions. Periportal hepatocytes receive high levels of nutrients, oxygen, and hormones, and they are specialized for oxidative function, gluconeogenesis, FA β-oxidation, and cholesterol synthesis. Conversely, perivenous hepatocytes obtain low levels of nutrients, oxygen, and hormones and function in glycolysis, lipogenesis, and drug detoxification.

FIGURE 3

The signaling pathway in hepatocytes during NAFLD progression. FFAs are transported through CD36. FFAs enter cells via the FA transporter CD36. Excessive FAs cause mitochondrial dysfunction either directly or indirectly through ER–stress. Increased ROS production activates the JNK signaling pathway, which causes apoptosis through activation of the proapoptotic Bcl-2 protein Bax and suppression of the anti-apoptotic Bcl-2 family proteins. As a result, damaged or dying hepatocytes can release DAMPs to activate Kupffer cells. Activated Kupffer cells can secrete large amounts of inflammatory and profibrotic cytokines. Consequently, they can activate HSC, leading to liver fibrosis. In normal hepatocytes, increased ROS activate AMPK, which maintains intracellular homeostasis through PGC1α and SIRT1. Activated PGC1α can inhibit ROS production through anti-oxidant mechanisms. On the other hand, during NAFLD, FFAs accumulate intracellularly and can inhibit autophagy through AMPK inhibition and mTORC1 activation. In fatty liver, upregulated CD36 in hepatocytes inhibits autophagy initiation through the AMPK/ULK1/Beclin1 pathway. In addition, mTORC1 activation in NAFLD inactivates the autophagy enhancer Pacer, thereby interfering with autophagosome–lysosome fusion. As another key factor that can regulate autophagy flux, cytoplasmic Ca²⁺ activates AMPK through CaMMK2 in normal conditions. Conversely, excessively increased cytoplasmic Ca²⁺ during NAFLD disrupts autophagy flux. During NASH progression, YAP/TAZ in hepatocytes can be hyperactivated. Dietary cholesterol promotes ER-mediated Ca²⁺ secretion and activated Ca²⁺-RhoA pathway can sequentially stabilize TAZ. TAZ entering the nucleus strongly induces the expression of genes involved in HSC activation and fibrosis. Abbreviations: CCL5, C-C motif chemokine ligand 5; CTGF, connective tissue growth factor; IL-1β, interleukin-1β; PDGF, platelet-derived growth factor; TGF-β, transforming growth factor-β; TNF-α, tumor necrosis factor-α.

FIGURE 4

Heterogeneity of intrahepatic biliary epithelial cells during NAFLD progression. Ductal plate cells consist of cholangiocytes, canal of hering, and hepatocytes. Cholangiocytes are fundamentally divided into large cholangiocytes and small cholangiocytes based on the diameter of the bile duct (BD). Large cholangiocytes depend on cAMP-signaling and are more susceptible to damage than small cholangiocytes. During liver injury, large cholangiocytes enter a senescence-associated secretory phenotype (SASP) state and secrete proinflammatory factors that exacerbate the damage. When the large cholangiocytes are damaged, small cholangiocytes act as a progenitor to large cholangiocytes and differentiate into large cholangiocytes via Ca²⁺-activated signaling. In a healthy state, quiescent cholangiocytes play an important role in immune and antimicrobial defense in response to PAMPs (e.g., LPS) originating from the intestine or DAMPs derived from damaged hepatocytes. PAMPs and DAMPs bind to TLR4 expressed by biliary epithelial cells. Quiescent cholangiocytes can also present antigens to unconventional T-cells including NKT cells and MAIT cells. During NAFLD progression, activated cholangiocytes contribute to inflammation and fibosis. The process is as follows: ^① During NAFLD progression, excessive FAs stimulate cholangiocytes. ^② Activated cholangiocytes, also known as reactive ductular cells (RDCs), show 1) proliferation phenotype or undergo 2) senescence. Senescent RDCs secrete SASP factors (TGF-β, PDGF, TNF-α and IL-1β). ^③ SASP factors stimulate immune cells and myofibroblasts, which promote inflammation and fibrosis, and some immune cells infiltrate into the bile duct. RDCs and mesenchymal and immune infiltration constitute ductular reaction (DR), a state in which they coexist. ^④ The recruitment and infiltration of immune cells into the bile duct cause 1) severe inflammation and 2) fibrosis. ^⑤ Small cholangiocytes transdifferentiate into large cholangiocytes to replenish the damaged cholangiocytes.

FIGURE 5

The signaling pathway in HSCs during NAFLD progression. TGF-β is well-known as a critical factor for HSC activation and liver fibrosis. The TGF-β signaling pathway is divided into a canonical SMAD-dependent pathway and a noncanonical SMAD-independent pathway. In a canonical SMAD-dependent pathway, TGF-β binds to and phosphorylates types I and II serine/threonine kinase receptors. Sequentially, it phosphorylates SMAD2/3 to form a complex with SMAD4. These SMAD complexes translocate into the nucleus, and regulates the expression of genes involved in oxidative stress and liver fibrosis. Both TGF-β and PDGF can also phosphorylate SMAD2/3 via JNK, leading to induction of fibrogenic genes. Conversely, anti-fibrotic SMAD7 can inhibit SMAD2/3 phosphorylation by directly binding to or indirectly degradation of TGF-β receptors. On the other hand, the SMAD2/3/4 complexes in nucleus can also induce NOX4, a major source of mitochondrial oxidative stress. NOX4-derived ROS production can increase TGF-β expression and secretion. Nrf2 is a transcription factor that mitigates ROS by increasing the expression of various ROS-detoxifying enzymes such as GPX2 and GST. On the other hand, TGF-β can also regulate various intracellular pathways, including PI3K/AKT, mTOR, MAPK, and Rho/GTPase, through a noncanonical SMAD-independent pathways. The Hh signaling pathway in HSCs is one of the important signaling pathways induced during NAFLD and NASH. During liver damage, Hh ligands are increased and then activate the Gli-responsive transcription factor to promote the expression of fibrogenic genes. Consequentially, qHSCs (qHSC markers: LRAT, BAMBI) transdifferentiate into aHSCs (aHSC markers: α-SMA, GFAP, PDGFR-β, COL1α1, COL1α3, desmin, and vimentin).

FIGURE 6

The signaling pathway of Kupffer cells during NAFLD progression. Kupffer cells can be activated by DAMPs released from damaged hepatocytes and LPS secreted by intestine during NAFLD progression. DAMPs and LPS bind to TLR4 on Kupffer cell membrane and activates TLR4 signaling. TLR4-mediate signaling pathways in Kupffer cells are divided into MyD88-dependent or MyD88-independent signaling. MyD88-dependent signaling is mediated by MyD88 and Mal/TIRAP, which activate MAPK signaling, including ERK, JNK, and p38, and activate NF-κB and AP-1 to produce pro-inflammatory cytokines. On the other hand, the MyD88-independent signaling is mediated by TRIF and TRAM, leading to the activation of IRF3 and regulates the expression of IFN. LPS can also promote NOX2 to produce ROS via TLR4 activation. Excessive ROS production by mitochondria promotes NF-κB and MAPK signaling to secrete pro-inflammatory cytokines. Conversely, UCP2, a mitochondrial inner membrane protein, counteracts ROS production to maintain homeostasis. Abbreviation: AP-1, activator protein 1; ETC., mitochondrial electron transport chain; IRF3, interferon regulatory factor 3; Mal/TIRAP, MyD88 adaptor-like/TIR domain containing adaptor protein; MAPK, mitogen-activated protein kinase; MyD88, myeloid differentiation primary response 88; NF-κB, Nuclear factor kappa B; NOX2, NADPH oxidase 2; TRAM, TRIF adaptor-related adaptor molecule; TRIF, Toll/IL-1R domain-containing adaptor-inducing IFN-β; ROS, Reactive oxygen species; UCP2, uncoupling protein 2.