Liquid Biopsy for the Diagnosis of Viral Hepatitis, Fatty Liver Steatosis, and Alcoholic Liver Diseases

During the progression from hepatitis to fibrosis, cirrhosis, and liver failure, the accumulation of stressed/damaged hepatocyte elements associated with liver inflammation is critical. The causes of hepatocyte injuries include viral hepatitis infections, alcoholic hepatitis, and non-alcoholic fatty liver disease. Hepatocyte-derived extracellular vesicles (Hep-EVs) released from stressed/damaged hepatocytes are partly responsible for liver disease progression and liver damage because they activate non-parenchymal cells and infiltrate inflammatory cells within the liver, which are in turn are an important source of EVs. This cell-to-cell signaling is prevalent during inflammation in many liver diseases. Accordingly, special emphasis should be placed on liquid biopsy methods for the long-term monitoring of chronic liver diseases. In the present review, we have highlighted various aspects of current liquid biopsy research into chronic liver diseases. We have also reviewed recent progress on liquid biopsies that focus on cell-free DNA (cfDNA), long non-coding RNA (lncRNA), and the proteins in EVs as potential diagnostic tools and novel therapeutic targets in patients with viral hepatitis, fatty liver steatosis, and alcoholic liver diseases.


Introduction
Chronic liver disease is a major public health issue globally. It includes chronic viral hepatitis, fatty liver disease, among other causes, and accounts for approximately 3.5% of deaths [1]. Viral hepatitis is a persistent state of liver inflammation caused by various viruses. It increases the risk of cirrhosis and hepatocellular carcinoma (HCC). Chronic hepatitis B virus (CHB) and chronic hepatitis C virus (CHC) infections are major risk factors for HCC, and the association has ranged from 45 to 80% in various regional epidemics. In recent decades, non-alcoholic fatty liver disease (NAFLD) and alcohol-associated liver disease have emerged as urgent etiologies associated with an increasingly westernized global lifestyle. It is worth noting that the increase in non-alcoholic steatohepatitis (NASH) associated with obesity and metabolic syndrome may have led, directly or indirectly, to an increase in

Liquid Biopsies Associated Viral Hepatitis Immunity
EVs also allow HCV to evade immunity by protecting the HCV RNA from degradation. The hepatitis A virus evades the human immune response by becoming encapsulated in the membranes of the host immune cells [29]. HCV-stimulated monocytes differentiate into polarized M2 macrophages; this activates HSCs and stimulates the secretion of EVs from HCV-infected hepatocytes [30]. Liver injury may stimulate the differentiation of monocytes into macrophages and hepatic macrophage recruitment. Elevated numbers of platelet-derived EVs are also associated with liver fibrosis biomarkers, such as serum hyaluronate and the N-terminal propeptide of type III procollagen [31]. Patients with chronic HCV infection experience greater platelet activation and increased levels of circulating platelet-derived EVs compared to those with chronic HBV infection. Immune cellderived circulating EVs are associated with advanced liver disease and may be identified by microparticle markers of cell type: T cells by CD4+/8+; monocytes by CD14+; neutrophils by CD15+; platelets by CD41+; and invariant natural killer T (iNKT) cells by Valpha24/Vbeta11 [32]. CHC patients can be identified among chronic hepatitis patients by cell-derived EV biomarkers. The authors of another study found that liver cells exposed to IFN-α conferred resistance to HBV replication via cell-to-cell communication through EVs in infected liver cells [33]. The immune cells are implicated in liver inflammation and liver-related diseases by host immune system regulation and alteration of the microenvironment [34].

Biomarkers of Liquid Biopsies Associated Viral Hepatitis
Nucleic acids and viral hepatitis-associated proteins have been identified in EVs from the sera of patients with chronic viral infections [35]. Long non-coding RNAs (lncRNAs) can affect the regulation of gene expression and have an impact on many different cellular processes. Xu et al. demonstrated significantly higher levels of exosomal HNRNPH1 in HBV-associated HCC patients with vascular invasion and lymph node metastasis than those in non-HCC patients [36]. A positive correlation among lncRNA-HULC, lncRNA-HEIH, and hepatitis B X-interacting protein (HBXIP) indicated that hepatitis B X protein (HBx) may alter lncRNA expression, which in turn may promote

Liquid Biopsies Associated Viral Hepatitis Immunity
EVs also allow HCV to evade immunity by protecting the HCV RNA from degradation. The hepatitis A virus evades the human immune response by becoming encapsulated in the membranes of the host immune cells [29]. HCV-stimulated monocytes differentiate into polarized M2 macrophages; this activates HSCs and stimulates the secretion of EVs from HCV-infected hepatocytes [30]. Liver injury may stimulate the differentiation of monocytes into macrophages and hepatic macrophage recruitment. Elevated numbers of platelet-derived EVs are also associated with liver fibrosis biomarkers, such as serum hyaluronate and the N-terminal propeptide of type III procollagen [31]. Patients with chronic HCV infection experience greater platelet activation and increased levels of circulating platelet-derived EVs compared to those with chronic HBV infection. Immune cell-derived circulating EVs are associated with advanced liver disease and may be identified by microparticle markers of cell type: T cells by CD4+/8+; monocytes by CD14+; neutrophils by CD15+; platelets by CD41+; and invariant natural killer T (iNKT) cells by Valpha24/Vbeta11 [32]. CHC patients can be identified among chronic hepatitis patients by cell-derived EV biomarkers. The authors of another study found that liver cells exposed to IFN-α conferred resistance to HBV replication via cell-to-cell communication through EVs in infected liver cells [33]. The immune cells are implicated in liver inflammation and liver-related diseases by host immune system regulation and alteration of the microenvironment [34].

Biomarkers of Liquid Biopsies Associated Viral Hepatitis
Nucleic acids and viral hepatitis-associated proteins have been identified in EVs from the sera of patients with chronic viral infections [35]. Long non-coding RNAs (lncRNAs) can affect the regulation of gene expression and have an impact on many different cellular processes. Xu et al. demonstrated significantly higher levels of exosomal HNRNPH1 in HBV-associated HCC patients with vascular invasion and lymph node metastasis than those in non-HCC patients [36]. A positive correlation among lncRNA-HULC, lncRNA-HEIH, and hepatitis B X-interacting protein (HBXIP) indicated that hepatitis B X protein (HBx) may alter lncRNA expression, which in turn may promote HCC development [37].
lncRNA-HULC modulates microRNA-539 (miR-539), which downregulates APOBEC3B, thereby promoting HBV replication [38]. Both serum and exosomal lncRNA-HEIH expression levels increase in HCV-related HCC patients, whereas serum lncRNA-HEIH expression levels are significantly lower than exosomal lncRNA-HEIH expression levels in CHC patients [39]. These data suggest that lncRNA is a potential liquid biotarget for HBV-related HCC. MHC class I chain-related A (MICA) is another HCV-associated liquid biopsy target. High levels of soluble MICA (sMICA) have been identified in the sera of HCV-induced HCC patients bearing the G allele as opposed to the A allele as a result of the single nucleotide polymorphism (SNP) rs2596538 [40]. There was an increase in the risk of liver fibrosis in the CHC patients with the G allele, whereas the levels of sMICA were elevated in the HCV-associated HCC patients following viral eradication [41,42]. A genome-wide association study (GWAS) demonstrated that elevated serum levels of MICA and soluble MICA (sMICA)-encoded by the G allele resulting from SNP rs2596542-increased the risk of HBV-associated HCC [43].
Viral amplification and replication could be used in target detection. Chronic HBV infection is characterized by a persistent episomal viral genome, namely covalently closed circular DNA (cccDNA). cccDNA constitutes a stable mini-chromosome within the infected hepatocyte nucleus [44]. The persistence of cccDNA and the inability of the immune system to eradicate the virus contribute to the failure of viral clearance and to relapse, even after treatment goals have been achieved. Therefore, true HBV clearance or functional cure is generally defined as the complete elimination of cccDNA from infected hepatocytes [44]. Theoretically, even if one molecule of HBV cccDNA with replication potential remains in a liver cell, there is still a risk of virological recurrence after ceasing antiviral therapy. The presence of pre-genomic RNA (pgRNA) in mature HBV viral particle nucleocapsids is linked to the persistence of viral infection and the risk of virological recurrence. Lin et al. demonstrated the use of real-time quantitative polymerase chain reaction (qPCR) to determine HBV pgRNA levels in the sera of HBV-infected patients, and to make a better assessment of changes in the sustained viral response (SVR) [45]. Regarding pathogenic cell-free genomics, Tokuhisa et al. were the first to demonstrate that the levels of circulating cfDNA increase in the sera of patients with HCV-related HCC [46]. Iida et al. also reported an association between the level of cfDNA in the serum and the metastatic capability of HCV-related HCC, which suggests the potential of cfDNA as an active metastasis biomarker following curative surgery [47]. Patients with CHC-related HCC have 3-4 times higher levels of cfDNA in their sera or plasma than their non-HCC CHC counterparts, and the level is 20 times higher than in healthy controls [48]. The viral hepatitis-related liquid biomarkers are listed in Table 1.

Biomarkers Associated Non-Alcoholic Steatohepatitis/Non-Alcoholic Fatty Liver Disease (NASH/NAFLD)
NAFLD is a well-known liver disorder, with 25% prevalence across the globe. It is broadly distributed in diverse populations and regions. NASH is the severe form of NAFLD, which is characterized by lobular inflammation or the chronic low-grade proinflammatory state of hepatocytes with or without fibrosis. NAFLD is currently the most prevalent cause of liver transplantation [49]. The prevalence of NAFLD has increased rapidly over the past few decades in the Asia-Pacific region, at a comparable rate to westernization. NAFLD is an intermediate stage in various progressive diseases that are not associated with high alcohol intake. Clinical manifestations include confined intrahepatic triglyceride accumulation (exceeding 5%) and necroinflammation of the hepatocytes, with some patients progressing to fibrosis, cirrhosis, and HCC. Lipotoxicity develops during the progression of NAFLD owing to the accumulation of toxic lipids and stressed/damaged hepatocytes; it is linked to metabolic disorders including dyslipidemia, type 2 diabetes, and obesity [50].
Various mechanisms contribute to the intrahepatic accumulation of fat. These include fluctuations in the level of fatty acids, increased lipogenesis, the secretion of very-low-density lipoprotein (VLDL), and suppressed clearance through β-oxidation [51]. NASH is characterized by an increase in the level of serum triacylglycerols (TAGs), and a positive hepatic histological index distinguishes NASH in NAFLD patients [52]. Minimal TAG levels may be hepato-protective, whereas prolonged lipid storage may escalate into inflammation and metabolic dysfunction [53]. The accumulation of TAGs-which occur predominantly as macrovesicular lipid droplets in hepatocytes-is associated with liver injury or necroinflammation [54]. One study using plasma samples demonstrated that peroxisome proliferator-activated receptor gamma (PPARγ) DNA methylation is associated with the fibrosis index and may be used as a severity predictor in NAFLD diagnoses [55]. The authors of another study found that homocysteine (Hcy) serum concentrations are significantly higher in NAFLD patients and are not influenced by chronic hepatitis [56]. Furthermore, there is a positive correlation between plasma Hcy levels and the severity of steatosis; therefore, plasma Hcy levels could be used to discriminate between NASH and simple steatosis [57]. Glutamine, glycine and serine which are precursors of antioxidants such as glutathione (GSH) were demonstrated to be decreases in the NAFLD patients compared to healthy individuals [58]. Proline and hydroxyproline, the elements of collagen protein, also observed to be elevated in serum of patients with NAFLD compared to healthy individuals [59]. Another study indicated that a rate-limiting enzyme Acetyl-CoA carboxylase (ACC) increased expression in NAFLD animal models [60]. Indeed, Alkaline phosphatase (ALKP) elevated levels are associated with hepatic fibrosis in steatohepatitis patients. ALKP serum level are notably higher in NASH patients compared to those without NASH [61].

Hepatocyte-Derived Vesicles Associated Non-Alcoholic Steatohepatitis/Non-Alcoholic Fatty Liver Disease (NASH/NAFLD)
With regards to NAFLD, EVs play a key role in the mechanism of liver damage and disease progression through the accumulation of lipotoxic lipids in hepatocytes. In turn, hepatocyte-derived extracellular vesicles (Hep-EVs)-which are discharged from damaged or stressed hepatic cells-exacerbate the progression of liver disease through the stimulation of non-parenchymal cells [62]. In mouse hepatocytes and human hepatocyte-derived carcinoma cell (HuH) models, palmitate and lysophosphatidylcholine (LPC) increase the release of EVs, demonstrating the conservation of toxic-lipids and cytochrome P450 2E1 (CYP2E1) within serum EVs. LPC-induced hepatic lipotoxicity is triggered by chemokine (C-X-C motif) ligand 10 (CXCL10), which is enriched in hepatocyte-derived vesicles, and promotes macrophage chemostasis [63]. This suggests that hepatocyte-derived EVs are potential biomarkers of NASH [64]. In addition, lipotoxic EVs from hepatocytes stimulate pro-fibrogenesis in hepatic stellate cells [59]. With regard to NASH, lipids facilitate the release of EVs through the tumor necrosis factor receptor superfamily member 10B (TNFRSF10B) signaling pathway in human and mouse hepatocytes [65]. The study demonstrated that the upregulation of toll-like receptor 9 (TLR9) pathway was activated by the high levels of hepatocyte mtDNA in the MPs of mouse plasma to induces lipotoxicity and inflammation in NASH patients [66].

Liquid Biopsies Associated Chemoattraction in Non-Alcoholic Steatohepatitis/Non-Alcoholic Fatty Liver Disease (NASH/NAFLD)
EVs play a role in cell-to-cell communication by transferring biologically active molecules into target cells while regulating NASH pathogenesis and progression. The fatty liver disease-related liquid biomarkers are listed in Table 2. Macrophage activation and influx in the liver are important for the progression of NAFLD because hepatic macrophages promote NASH development via cytokines IL-1, IL-6, and TNFα [67]. A model comprising Mlk3−/− mice fed a diet that was high in fats and carbohydrates exhibited suppressed cytokine expression and macrophage infiltration compared to wild-type (WT) mice [68]. This indicates that non-parenchymal cells and infiltrated inflammatory cells are optional yet important sources of EVs that perpetuate liver injury. The TLR9 is facilitated by the chemotaxis of neutrophils and M1 macrophages-proves that TLR9 is a pro-inflammatory activator in NASH [69]. The production of Hep-EVs increases in hepatocytes treated with palmitic acid (PA), and hepatic stellate cells (HSCs) are stimulated when treated with exosomes from PA-treated hepatocytes in a cell culture model [70]. Exosomes released by healthy or compromised hepatocytes and Hep-EVs are crucial in cell-to-cell communication, especially in hepatocytes and hepatocyte-to-HSC signaling. EVs derived from hepatocytes containing excessive levels of lipids promote liver fibrosis through pro-inflammation, which directly affects immune cells. Kornek et al. reported increased numbers of EVs derived from invariant natural killer T cells and CD14+ monocytes-which are associated with alanine transaminase (ALT)-in plasma samples from NAFLD patients [32]. There is an increase in inflammatory cell-derived Hep-EVs in NAFLD patients and in patients with either or both of alcohol-related cirrhosis and chronic hepatitis C-related cirrhosis [32,71]. Immature myeloid cells (CD11b + , Ly6C hi , Ly6G -) have also been detected in a high-fat diet mouse model of NASH [72]. Furthermore, mesenchymal stem cell (MSC)-derived EVs are linked to liver inflammation and NASH progress, and are associated with increased proinflammatory markers (TNF-α, IL-1β, IL-6, and IL-12) [73]. Mouse obesity models have been used to demonstrate that levels of circulating adipocyte-derived microparticles are high in stressed adipocytes, which secrete chemoattraction "find-me" signals to recruit macrophages and monocytes [74]. Notably, palmitate-stimulated EVs are enriched in ceramide (C16:0). EVs chemoattract macrophages via ceramide-derived sphingosine 1 phosphate (S1P), which leads to macrophage activation [75]. This result also indicated that the amino acid metabolomics also can be detected by the liquid biopsies and significantly associated with the progression of liver fibrosis [76]. To date, the literature indicates that the EVs secreted by various cells play a crucial role in inflammatory regulation in advanced steatosis. However, the major components of EVs that are involved in cell or cytokine activation require further investigation. Figure 2 is an illustration of the NAFLD/NASH effects on the liver driven by lipid accumulation and their effects. Extracellular vesicles (EVs); cytochrome P450 2E1 (CYP2E1); mitochondrial DNA (mtDNA); non-alcoholic fatty liver disease (NAFLD); non-alcoholic steatohepatitis (NASH); peroxisome proliferator-activated receptor gamma (PPARγ); toll-like receptor 9 (TLR9); sphingosine 1 phosphate (S1P). homocysteine (Hcy); cytochrome P450 2E1 (CYP2E1); mitochondrial DNA (mtDNA); non-alcoholic fatty liver disease (NAFLD); non-alcoholic steatohepatitis (NASH); peroxisome proliferator-activated receptor gamma (PPARγ); toll-like receptor 9 (TLR9). Int. J. Mol. Sci. 2020, 21, x 9 of 21

Biomarkers Associated Alcoholic Liver Disease (ALD)
Alcoholic liver disease (ALD) foreshadows a wide variety of liver pathologies from basic steatosis to advanced liver injuries such as steatohepatitis and fibrosis/cirrhosis [77]. Steatosis almost always occurs in the hepatocytes of people that consume excessive amounts of alcohol. In 20-40% of heavy drinkers there is progression to steatohepatitis through neutrophil infiltration and related inflammation [78]. Excessive alcohol consumption promotes the production of prooxidants and strengthens the antioxidant mechanism, which leads to hepatocyte injury accompanied by inflammation; this eventually results in liver failure or cancer [79]. ALD hepatitis is directly related to the toxic effects of alcohol. It is indirectly caused by fecal infection or adipose tissue impairment resulting from bacteria-related endotoxins, including lipopolysaccharides (LPSs). It is also caused by elevated levels of free fatty acids, and by proinflammatory adipokines, which are found in the blood but also regulate liver function. Several authors have reported changes in the levels of ALD-corelated proteins in the serum. TG accumulation in the liver is the first histological change due to alcohol, and is reversible. Trinchet et al. (1994) reported that elevated levels of primary bile acids are associated with alcoholic hepatitis (AH) [80]. Moratti et al. reported that increased plasma levels of protein tyrosine phosphatase receptor type gamma (PTPRG) isoform sPTPRG in EVs excreted from the human hepatocellular carcinoma cell line HepG2 are associated with alcohol-related hepatic injury [81]. FSP27/CIDEC (fat-specific protein 27/cell death-inducing DNA fragmentation factor alpha-like effector c)-which promotes alcohol-stimulated liver damage-was highly expressed in the hepatocytes of a mice model following chronic and binge alcohol consumption, and in alcoholic hepatitis (AH) patients [82]. The summary of the liquid factors associated with liver inflammation in ALD as shown in Figure 3 in advance.
proteins in the serum. TG accumulation in the liver is the first histological change due to alcohol, and is reversible. Trinchet et al. (1994) reported that elevated levels of primary bile acids are associated with alcoholic hepatitis (AH) [80]. Moratti et al. reported that increased plasma levels of protein tyrosine phosphatase receptor type gamma (PTPRG) isoform sPTPRG in EVs excreted from the human hepatocellular carcinoma cell line HepG2 are associated with alcohol-related hepatic injury [81]. FSP27/CIDEC (fat-specific protein 27/cell death-inducing DNA fragmentation factor alpha-like effector c)-which promotes alcohol-stimulated liver damage-was highly expressed in the hepatocytes of a mice model following chronic and binge alcohol consumption, and in alcoholic hepatitis (AH) patients [82]. The summary of the liquid factors associated with liver inflammation in ALD as shown in Figure 3 in advance.

Hepatocyte-Derived Vesicles Associated Alcoholic Liver Disease (ALD)
Investigations into EVs as biomarkers for alcoholic hepatitis in humans have revealed increased levels of EVs in alcoholic hepatitis patients and patients who consume excessive amounts of alcohol

Hepatocyte-Derived Vesicles Associated Alcoholic Liver Disease (ALD)
Investigations into EVs as biomarkers for alcoholic hepatitis in humans have revealed increased levels of EVs in alcoholic hepatitis patients and patients who consume excessive amounts of alcohol [83]. A study involving alcohol-fed rodents and human patients who consumed alcohol revealed elevated levels of cytochrome P450-2E1 and cytochrome P450 isoforms in exosomes. The authors further reported an increase in the number of EVs involving CYP2E1 and P450 isoforms that was associated with an increase in oxidative stress in the endoplasmic reticulum [84]. Such endotoxins in the circulation increase the likelihood of progression to advanced-stage disease through fecal infection [85]. EV levels are elevated in patients with a history of alcohol abusive compared to those that consume extreme levels of alcohol and those that do not drink. EV levels are positively associated with ALT serum levels, mtDNA serum levels, and circulating neutrophil levels [86]. The injection of heat shock protein 90-containing EVs-which were isolated from the sera of alcohol-consuming mice-into naive mice demonstrated the internalization of injected EVs in hepatocytes; this resulted in elevated CCL2 expression [87], which indicates that alcohol activates an innate immune response through hepatocyte-derived EVs.

Liquid Biopsies of Markers Associated with Inflammation in Alcoholic Liver Disease (ALD)
The accumulation of hepatic macrophages has been observed in the portal tracts of ALD patients. EVs stimulate the activation of macrophage activation and the induction of inflammatory cytokines, as demonstrated in an unbiased microarray-based antibody neutralization experiment [92]. Alcohol-exposed, monocyte-derived EVs activate naive monocytes to polarize M2 macrophages by increasing the expression of surface receptors CD68, CD206, and CD163, and the secretion of IL-10 and TGFβ, which elevates phagocytic activity [89]. Furthermore, the levels of CD40L-rich EVs were elevated in the sera of AH patients. The proteins detected within EVs during macrophage activation suggests that CD40L may stimulate the activation of macrophages in experimental alcoholic hepatitis models [92]. Chemotaxis is the initiating step in neutrophil recruitment, and requires chemokines such as IL-8, which is essential for neutrophil recruitment [93]. IL-8 levels are extremely elevated in both circulating and hepatic neutrophils, which are associated with alcoholic hepatitis and disease aggression [94][95][96]. CXCL1 is an IL-8 homologue that has been demonstrated to promote the recruitment of neutrophils in a mice model. Studies in a mouse model of ALD have shown that elevated levels of CXCL1 in the serum and hepatocytes exacerbate liver injury [58,97]. In endothelial cell-derived extracellular vesicles, ethanol stimulates the upregulation of lncRNAs HOTAIR and MALAT1, which facilitates the pro-angiogenic effects of endothelial-derived vesicles [98]. The alcoholic hepatitis-related liquid biomarkers are listed in Table 3. Protein tyrosine phosphatase receptor gamma extracellular domain (sPTPRG); Alanine transaminase (ALT); alcoholic hepatitis (AH); alcoholic liver disease (ALD); alcoholic steatohepatitis (ASH); chemokine (C-C motif) ligand 2 (CCL2); chemokine (C-X-C motif) ligand 1 (CXCL1); extracellular vesicle (EV); Microvesicles (MVs); long non-coding RNA (lncRNA); microRNA (miR); mitochondrial DNA (mtDNA).

Conclusions
In the present review, we have highlighted the use of virus-related cfDNA, proteins and lncRNA in EVs/EPs and exosomes for liquid biopsies for the diagnosis of chronic liver diseases (Figure 4). Numerous studies have demonstrated that EVs affect the chronic inflammatory response in the progression of liver diseases. EVs released from stressed hepatocytes are fundamental modulators of these responses and take effect through various actions in different target cells including HSCs, MSCs, and macrophages; they are implicated in complex networks of cell-to-cell communication in liver disease development and progression. Some studies have presented analyses of EVs as carriers of specific proteins or genes involved in the biogenesis of diseases which can provide therapeutic targets in the future. This indicates the potential of the liquid biopsy as a non-invasive and accurate approach to the diagnosis and monitoring of chronic liver disease.  . The potential of the liquid biopsy as a non-invasive and accurate approach to the diagnosis and monitoring of chronic liver disease by disease associated markers from EVs, cfDNA, and serum protein. Biomarks have potential for further gene allele and mutation study to design the genomic testing depends on the gene expression and sequencing result for personalized therapeutic strategy. The biomarks expression testing level not only to monitor the disease process, also can apply for the drug design, drug testing and therapeutic efficiency in the future.