Survival Fate of Hepatic Stem/Progenitor and Immune Cells in a Liver Fibrosis/Cirrhosis Animal Model and Clinical Implications

Abstract

This chapter provides novel information about the survival features of hepatic resident stem/progenitor cells (NG2+ HSPs) during liver fibrosis/cirrhotic development. A well-defined diethylnitrosamine (DEN)-induced liver fibrosis/cirrhotic/cancer mouse model was developed to evaluate the fate of the HSPs and its clinical implications. This model possess three time-zones during the disease development: fibrosis (3–5 weeks post-DEN), cirrhosis (6–10 weeks post-DEN), and cancers (up to 10 weeks post-DEN). During this process, the model represents histological patterns similar to those described in humans and shows better survival of the HSPs in the fibrotic zone, which was correlated with inflammatory signals, as compared to the cirrhotic zone. It has also been discovered that immune CD8+ T cells in the fibrotic zone are beneficial in liver fibrosis resolution, suggesting that the fibrotic time zone is important for mobilizing endogenous HSPs and cell-based therapy. As such, we hypothesize that clinical strategies in fibrotic/cirrhotic liver treatment are necessary either in time at the fibrotic phase or to adopt an approach of regulating HSP viability when the disease develops into the cirrhotic phase.

1. Introduction

The healthy liver performs over 500 different functions to maintain its homeostasis [1, 2], and any assault could not only impact the architecture of hepatic cells [3] but also injure niche cells, like inflammatory and immune cells such as CD8-positive cytotoxic T lymphocyte subset (CD8⁺ T cells) which have been recently highlighted [4]. Liver cirrhosis is an end-stage liver disease (ESLD) and a major cause of morbidity and mortality globally [5]. It is often caused by acute liver injury or the progressive development of liver fibrosis or other chronic liver disease such as virus infection, etc. In China, the total number of patients with various types of chronic liver disease exceeds 300 million, and more than 1 million people die each year from ESLD [6]. According to the Centers for Disease Control (CDC), ESLD is the twelfth leading cause of death in the United States, with approximately 44,000 deaths per year [7]. For example, chronic hepatitis B virus (HBV) infection-mediated ESLD and inflammatory disease are global public health issues. It was estimated in 2017 that 257 million people live with chronic HBV infection, a prevalence of 3.5% [8], and there are 93 million people infected with HBV living in China [9]. Approximately 15–40% of patients with chronic HBV will develop liver cirrhosis, and 4–5% of patients may progress toward decompensated liver cirrhosis (DLC) [9], denoted in the chemical reagent diethylnitrosamine (DEN)-induced liver fibrosis/cirrhosis animal model at 6–10 weeks post-DEN in this chapter [9]. The 5-year mortality in patients with compensated liver cirrhosis (CLC), denoted as fibrotic phase (6–10 weeks post-DEN) in this chapter, is 14–20% and with DLC as high as 70–86% [5, 8, 9] which imposes a substantial health burden on many countries. It has been studied in animals that continuous activation of hepatic stellate cells (HSCs) leading to accumulation and over-deposition of the extracellular matrix (ECM) in the parenchymal liver compartment [10, 11] is a key mechanism for the liver from homeostasis to fibrosis (3–5 weeks post-DEN) to cirrhosis (6–10 weeks post-DEN) and eventually the outcomes of liver cancer like hepatocyte cellular carcinoma (HCC, >10 weeks post-DEN), which is the most common liver cancer (Figure 1A). In humans, there are generally four categories from healthy liver to HCC, including homeostasis, compensation liver cirrhosis phase (CLC), decompensation liver cirrhosis phase (DLC), and liver cancer (HCC) (Figure 1A). Of note, there are currently none of available treatments to specifically target other than liver transplantation. Unfortunately, there are simply not enough donated livers to meet the demands; therefore, research on nonsurgical strategies to prevent the development of chronic liver disease is urgently needed. Stem cell therapy is one of the options for treating liver fibrosis/cirrhosis [12] and can partially recover liver function [13, 14]. However, the problem is that clinical trials using stem cells to treat liver fibrosis/cirrhosis, especially for DLC treatment, seem not to be so successful [12, 15, 16, 17] because of the short-term survival of cells in DLC microenvironment [18].

Figure 1.

Application of DEN produces a relatively sharp line in different phases during the model disease development. (A) Evolution of ESLD during disease development. (B) Masson trichrome staining identified pathogenesis (blue, boxes) in hepatic sections at different phases of DEN-induced liver fibrosis-cirrhosis-cancers (HCC, an arrow) in a mouse model. Scale bar = 100 μm.

Therefore, if animal models with liver fibrosis/cirrhosis laboratory scores capture patients with the same disease, pathological features would be very meaningful for future clinical stem cell-based therapeutic strategies. As such, we have for the first time successfully set up a liver fibrosis/cirrhosis/cancer animal model in C57BL/6 mice in 2015, demonstrating histological patterns similar to those described in humans during the disease process [19]. Interestingly, this model shows that during the disease process, the resident HSPs (denoted as NG2⁺ HSPs) [18] respond to DEN-mediated inflammatory signals differently, resulting in different fates during liver fibrosis/cirrhosis development. More interestingly, immune CD8⁺ T cells have also been found recently to involve in improving liver fibrotic load [20]. In this chapter, we have provided novel information on the correlation of hepatic inflammatory activity with microenvironment resident HSPs (NG2⁺ HSPs) and how HSP survival fate are also related to the hepatic resident CD8⁺ T cells during the disease development on this model.

2. The chemical reagent (DEN)-induced liver fibrosis/cirrhosis and carcinogenesis in C57BL/6 inbred mice fed a drinking water diet

DEN is a nitrosamine-containing chemical reagent, and its toxic properties in mammals are well established [21, 22, 23]. Studies have shown that oral administration of the smallest quantities of DEN or dimethylnitrosamine (DMN) results in severe hepatic cell injury in both animals and humans [24, 25, 26, 27]. The most prominent chemical reagents are intense neutrophilic infiltration, extensive centrilobular hemorrhagic necrosis, bile duct proliferation, fibrosis, and bridging necrosis that end in hepatocarcinogenesis [28].

DEN was first brought to general attention in 1937 [29] when it was reported to be the causative agent that induces liver injury in men. This assumption was experimentally confirmed in 1954 by Barnes and Magee, who found that a single oral or parental dose (20–40 mg/kg body weight) of the reagent acts primarily as a liver poison producing severe liver necrosis in both small and large animals [30] of which the landmark experiment shows a sharp line of demarcation between the totally destroyed parenchyma and apparently uninjured liver cell areas in the model liver lesions [30]. Furthermore, the animal model shows that chronic application of the chemical agent in rats resulted in a high incidence of hepatic malignancy [31]. The extension of these studies has been subsequently revealed by many other researchers [30, 32, 33]. These findings suggest that the chemical and physical properties of DEN may be of interest for understanding the hepatic pathological characteristics underlying the evolution of liver fibrosis/cirrhosis/HCC formation [25], although the hepatotoxic and carcinogenic mechanism was not known until 1963 [34].

Moreover, although the application of DEN has become a well-established model in animals for studies of the pathogenetic alterations underlying the formation of liver fibrosis, cirrhosis and cancers, the single diet DEN for analyzing liver fibrosis/cirrhosis in wild-type mice has never been investigated. As such, applied C57BL/6 wild-type inbred mice with a single diet of 0.014% DEN in drinking water; 6 days/week for 15 weeks, we successfully induced a liver fibrosis/cirrhosis/cancer mouse model [19]. The model shows that DEN is extremely effective in inducing hepatic fibrosis [21], cirrhosis and cancers (Figure 1B) that are compatible with the deterioration of liver functions [22, 35].

3. Prolonged single-diet DEN administration within drinking water in C57BL/6 mice results in hepatic inflammation that impacts resident HSP fate

At present, although stem cell-based therapy as an emerging approach is widely studied for a variety of causes of CLF and CLC, for example, nonalcoholic fatty liver disease (NAFLD) [36], alcohol-associated liver disease (ALD) [37], and drug-induced liver injury (DILI) [38] etc.; however, due to the short-term survival of HSPs in the cirrhotic niche [18], the effect seems not to be so successful in the clinic.

It is known that inflammation is involved in pathological features during regenerative fibrotic nodule formation, which replaces normal functional liver parenchyma to remodel the vasculature in the liver and ultimately compromises liver function [39, 40, 41, 42]. Histologically, the inflammatory responses to toxic signals of DEN and influence fibrotic load have been characterized by our [18] and other studies [43]. These studies exhibit monocyte infiltration and collagen deposition accompanies varying degrees of deformation of liver structures identified by hematoxylin-eosin (H&E) and Masson’s trichrome staining, resulting in aggravated fiber load. Advanced fibrosis of the liver has been considered irreversible and is itself a risk factor for liver cancers such as HCC. Traditionally, it has often been thought that a beneficial way to prevent advanced fibrosis or liver cirrhosis is to control inflammation, while a recent study suggests that inflammation may be helpful for resident HSP activation and survival during hepatic disease development [18].

Several strategies to mobilize endogenous HSP-based therapies have been studied within the past few decades, but the underlying relationship between the HSP cells with their inflammatory niche signals in injured liver is largely unknown. We have currently demonstrated that prolonged oral water feeding with 0.014% DEN produces a sharp line between liver fibrosis (3–5 weeks post-DEN) and cirrhosis cancers (6–10 weeks post-DEN) [18]. Of note, in this model, we have found an interesting phenomenon, showing a positive correlation of resident HSP (NG2⁺ HSP) survival with hepatic inflammation in the fibrotic stage (3–5 weeks post-DEN) and when an irregular inflammatory response occurred in the later fibrosis or cirrhosis stage (3–5 weeks post-DEN), the HSP cells (NG2⁺ HSPs) died rapidly (Figure 2A and D), represented as gradually reduced Ki-67⁺ cells of NG2⁺ HSPs (Figure 2B) and increased terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL)-expression within the NG2⁺ HSPs (Figure 2C), suggesting that some unknown signals in the cirrhotic liver niche could trigger the HSP cell (NG2⁺ HSP) apoptosis [44].

Figure 2.

Correlation of hepatic inflammation with resident hepatic stem/progenitor cell (NG2⁺ HSP) activity. (A) H&E staining detected hepatic infiltrating mononuclear cells at the fibrotic phase (3–5 weeks post-DEN) and cirrhotic phase (7–9 weeks post-DEN) during DEN-induced liver development. Scale bar = 100 μm. (B) Using anti-NG2 (red) and Ki-67 (green) antibodies for hepatic resident NG2⁺ HSP cell proliferation at the fibrotic phase (3–5 weeks post-DEN) and cirrhotic phase (7–9 weeks post-DEN) (merged as brown, arrows) during DEN-induced liver development. Scale bar = 200 μm. (C) The TUNEL (green) assay identified hepatic resident NG2⁺ HSP cell (red) apoptosis (merged as brown, arrows) at the fibrotic phase (3–5 weeks post-DEN) and cirrhotic phase (7–9 weeks post-DEN) during DEN-induced liver development. Scale bar = 200 μm. (D) The International Simplified Grading and Staging System (ISGSS) was used for inflammation index activity and immunofluorescence staining for inflammation degree (denoted as G, pink) and NG2⁺ HSP activity (brown) evaluation. ^*#p < 0.05 vs. fibrotic phase, with Student’s t test.

4. The 0.014% DEN-treated liver fibrosis/cirrhosis mouse model-associated immune role of resident hepatic CD8⁺ T cells

The DEN-induced dynamic liver disease mouse model lining hepatic homeostasis, fibrosis, cirrhotic and HCC phases seems similar to humans. Clinically, liver cirrhosis comprises two stages as abovementioned: the fibrotic phase, often asymptomatic, also known as a compensated stage (CLC), and the cirrhotic phase or decompensated stage, characterized by complications arising from portal hypertension and hepatic insufficiency (DLC) [45, 46]. The DLC is also considered a systemic disease because it affects most organs and systems of the body, including the immune system [47, 48].

On the topic of fibrotic/cirrhotic liver disease and immune correlation, we would like to mention the latest idea termed cirrhosis-associated immune dysfunction (CAID) [49, 50], characterized by two key components: systemic inflammation and immune deficiency. It shows variable intensity depending on the stage of liver fibrosis/cirrhosis and the presence of incidental events. With this, we would like to give a better representative example of cirrhosis-mediated liver failure, acute-on-chronic liver failure (ACLF), a syndrome characterized by acute decompensation of cirrhosis, hepatic and/or extrahepatic organ failures and high short-term mortality [51], as well as the most severe immune alterations in the patients with this disease [51]. Patients with liver cirrhosis who develop these stages/diseases combine the highest grade of systemic inflammation [41] and severe immunodeficiency [52], which leads not only to an increased risk of infections but also to pathogenic liver organ failure [53] or cancers (HCC) [54, 55]; however, how these phenomena underlie biological substrates is unclear. One study shows for the first time that depression or anxiety grade of patients with liver cirrhosis is correlated with CD8⁺ T-cell signs [56], suggesting that an imbalance of CD8⁺ T cells may be a factor facilitating neurological disease patients with cirrhotic liver disease.

Although at present it is considered that advanced liver fibrosis to be irreversible, recent clinical evidence demonstrating substantial fibrosis resolution following different successful treatments overturned this dogma [57]. Accordingly, significant efforts have been made to inhibit advanced liver fibrosis through a variety of modulations, such as targeting specific chemokines [58], cytokines [59], and even immune CD8⁺ T cells [60]. However, both pathogenic and suppression properties of intrahepatic CD8⁺ T cells have been highlighted in liver fibrosis/cirrhosis progression [61], and the functional and phenotypical characteristics of the CD8⁺ T cells associated with positive benefit in the liver disease are largely unknown. Albillos et al. [20] used murine diet-induced nonalcoholic steatohepatitis model (NASH) [2], a currently the leading cause of chronic liver disease worldwide [62], and mainly characterized lobular inflammation and hepatocyte ballooning [63], demonstrating a direct role of CD8⁺ T cells in fibrosis resolution by promoting HSC apoptosis in a CCR5-dependent manner [63], primarily highlighting the undefined role of liver-resident CD8⁺ T cells in the fibrotic/cirrhotic liver niche.

Also, in a recent preliminary study, tracking CD8⁺ T cells with immunofluorescence staining in the DEN mouse model to examine the dynamic changes of intrahepatic CD8⁺ T cells during the model disease development, we curiously found that CD8⁺ T cells were relatively higher in the fibrotic phase (3–5 weeks post-DEN) compared to their in the cirrhotic phase (3–5 weeks post-DEN) where dropping after 7 weeks post-DEN (Figure 3A and Ba,b), supporting that hepatic resident CD8⁺ T cells may have beneficial role for liver fibrosis/cirrhosis [20], maybe hinting a previously unappreciated role of hepatic resident CD8⁺ T cells in promoting injured liver tissue repair, need to be further investigated.

Figure 3.

Dynamic changes in hepatic resident CD8⁺ T cells during DEN-induced liver fibrosis/cirrhosis development. (A) The expression of hepatic resident CD8⁺ T cells was higher in the fibrotic phase (3–5 weeks post-DEN) than in the cirrhotic phase (7–9 weeks post-DEN). Scale bar = 200 μm. (Ba, b) Hepatic resident CD8⁺ T cells dramatically increased at 4 weeks post-DEN (a) and sharply decreased at 8 weeks post-DEN (b) during model disease development. ^*p < 0.05, 8 vs. 4 weeks, with Student’s t test.

5. Concluding remarks

This chapter collectively delivers information about the DEN-induced liver fibrosis/cirrhosis in a small amount (0.014%) of water drinking diet chemical (DEN)-induced hepatic fibrosis, cirrhosis and carcinogenesis mouse model and clinical implications, and provides additional insights into the liver possibility of the positive effect of CD8⁺ T cells in the animal model-associated pathogenesis. The sequence of pathophysiological alterations in the model has a high similarity to what happens in humans. In comparison to other mouse models, the DEN-induced liver fibrosis-cirrhosis-HCC model on a natural genetic background C57BL/6 inbred mice sounds favorable, and prolonged application of DEN to induce hepatic inflammatory response and immune CD8⁺ T cell activation after a period of latency are beneficial for endogenous HSP cells (NG2⁺ HSP) mobilization and survival. Based on our experience and on a multitude of independent studies in the field, we recommend that for treatment strategies not inhibit but regulate inflammation and immune CD8⁺ T-cells during liver fibrosis/cirrhosis development. Alternatively, the use of male mice at middle age (approximately 8–10 weeks old) and body weight of the animals should be regularly measured and documented (approximately 22–25 g), and the extent of pathological features (evolution from homeostasis to fibrosis to cirrhosis to HCC formation) should always be histologically proven and documented. Further studies are warranted to identify the specific signaling or molecules that contribute to the development of hepatic homeostasis to fibrosis, then cirrhosis and finally cancer (HCC) in the liver.

Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China (81873586) and the Army Medical University of China (2021-20180-52).

Conflict of interest

The authors declare no conflicts of interest.

Abbreviations

ACLF	acute-on-chronic liver failure
ALD	alcohol-associated liver disease
CDC	Centers for Disease Control
DEN	diethylnitrosamine
DILI	drug-induced liver injury
DLC	decompensated liver cirrhosis
ESLD	end-stage liver disease
HBV	hepatitis B virus
HCC	hepatocyte cellular carcinoma
H&E	hematoxylin-eosin
HSCs	hepatic stellate cells
NAFLD	nonalcoholic fatty liver disease
NG2-HSPs	neuroglia antigen 2-expressing hepatic stem/progenitor cells
TUNEL	terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling