**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 [39–42]. Histologically, the inflammatory responses to toxic signals of DEN and influence fibrotic load have been characterized by our [18] and other studies [43]. These

#### **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<sup>+</sup> 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<sup>+</sup> 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.*

#### *Survival Fate of Hepatic Stem/Progenitor and Immune Cells in a Liver Fibrosis/Cirrhosis… DOI: http://dx.doi.org/10.5772/intechopen.106220*

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<sup>+</sup> 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<sup>+</sup> HSPs) died rapidly (**Figure 2A** and **D**), represented as gradually reduced Ki-67<sup>+</sup> cells of NG2<sup>+</sup> HSPs (**Figure 2B**) and increased terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL)-expression within the NG2<sup>+</sup> HSPs (**Figure 2C**), suggesting that some unknown signals in the cirrhotic liver niche could trigger the HSP cell (NG2<sup>+</sup> HSP) apoptosis [44].

#### **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

**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<sup>+</sup> 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.*

an imbalance of CD8<sup>+</sup> 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<sup>+</sup> 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<sup>+</sup> 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)

*Survival Fate of Hepatic Stem/Progenitor and Immune Cells in a Liver Fibrosis/Cirrhosis… DOI: http://dx.doi.org/10.5772/intechopen.106220*

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<sup>+</sup> T cells in promoting injured liver tissue repair, need to be further investigated.
