1. Introduction
Fatty liver disease is the most common chronic liver disease worldwide, which includes alcoholic fatty liver disease (AFLD) and nonalcoholic fatty liver disease (NAFLD), with the prevalence of 6% and 23.7% in Europe [
1], respectively. NAFLD is driven by metabolism syndrome, associated with obesity, insulin resistance, and hyperlipidemia [
2], and is projected to increase by up to 56% between 2016 and 2030 in several European countries [
3]. NAFLD is a systemic inflammatory disease, and it can develop from fat accumulation (simple steatosis) to steatohepatitis and even advanced diseases, such as cirrhosis and hepatocellular carcinoma (HCC).
Hepatitis C virus (HCV) is a hepatotropic virus, causing both acute and chronic infection in human beings. It is estimated that 55–85% of individuals with HCV infection will develop into a persistent infection [
4]. HCV infection is closely associated with lipid metabolism defects throughout the viral lifecycle [
5]. The prevalence of hepatic steatosis in chronic HCV infection ranges from 50 to 80%, 2.5-fold higher than in the general population and in the other forms of chronic liver disease [
6]. Two main types of steatoses in patients with HCV infection have been defined: the metabolic type of steatosis associated with a high body mass index (BMI), hyperlipidemia and insulin resistance; and the virally induced steatosis that is linked with HCV genotype 3 infection [
7]. Over the past few years, numerous direct-acting antiviral (DAA) agents have been used in patients with HCV infection with high efficacy and safety; however, the steatosis disorder remains in patients with HCV after successful virus clearance, especially for the metabolic type of steatosis [
8,
9]. Moreover, obesity, diabetes, liver cirrhosis, alcohol consumption, and endpoints achieving sustained viral response (SVR) are independently associated with persistently increased liver enzymes after HCV clearance [
9]. Studies that compare the non-invasive method of measuring hepatic steatosis, controlled attenuation parameter (CAP), to the golden standard, liver biopsy, have illustrated that CAP has high diagnostic accuracy for detecting hepatic steatosis [
10,
11,
12,
13]; however, studies evaluating steatosis and fibrosis in liver biopsies after SVR are still lacking [
14].
HCV infections are often accompanied with alteration in the hepatic and systemic inflammatory state, characterized by the upregulation of several soluble mediators. Several studies have investigated the inflammatory cytokines and chemokine milieu in both acute [
15,
16] and chronic HCV infection [
17,
18]. Acute and chronic HCV infection significantly disrupts the milieu of soluble inflammatory mediators (SIMs) [
18,
19]; however, DAA-induced HCV clearance is not able to completely restore the altered SIMs [
18,
19]. Interestingly, it has also been reported that DAA treatment is followed by a substantial weight gain in one-third of patients during long-term follow-up [
20]. Previous data have shown that some soluble mediators can regulate the development of steatosis, such as soluble CD163 [
21], monocyte chemoattractant protein (MCP)-1, interleukin (IL)-8, and IL-6 [
22]. Therefore, it is of potential interest to investigate whether SIMs are associated with the steatosis status in patients with chronic HCV infection after virus clearance.
In the present study, we made use of a well-characterized cohort of patients with chronic HCV infection who were cured with DAA. By comparing the levels of SIMs in patients with different grades of steatosis at long-term follow up (96 w) after viral clearance, we may identify markers as indicators for steatosis.
4. Discussion
In this study, we comprehensively analyzed the expression of SIMs in patients with chronic HCV infection in relation to the status of CAP values at the long-term follow up after the virus clearance. Overall, our data suggested that four markers, SCF, TWEAK, FGF-21, and IL-18R1 differed in the different status of CAP value at 96 w after virus clearance, and these four markers were all significantly correlated with the CAP value.
A previous study demonstrated that hepatic steatosis is common in Egyptian HCV patients, and it increases after HCV eradication with DAAs [
25]. In line with this, our study also illustrated that steatosis disorder remained in most patients despite successful HCV clearance. In fact, the metabolic type of steatosis in HCV patients, except for genotype 3, is mainly linked to factors such as a high BMI, hyperlipidemia, and insulin resistance [
26,
27], similar to HCV-uninfected individuals. Moreover, an unexpected elevation in hepatic steatosis after DAA treatment has been found to be linked with lipid storm, elevation of LDL, and sdLDL [
28]. Consistently, our study also found elevated total cholesterol, triglycerides, and LDL in patients at 96 w compared to those at baseline (
Supplementary Figure S1), which may have contributed to the increased hepatic steatosis; however, the reasons for the increased total cholesterol, triglycerides, and LDL after HCV eradication still need to be elucidated.
At 96 w, we observed four markers that differed among different status of CAP values. TWEAK has been reported to reduce lipid accumulation in human liver cells, and the decreased soluble TWEAK concentrations are independently associated with the presence of NAFLD [
29]. However, the overexpression of FGF-21 ameliorates obesity and liver steatosis [
30]. Consistently, in our study, the expression of TWEAK was lower in patients with CAP > 299 dB/m, while the expression of FGF-21 was higher in patients with CAP > 299 dB/m. Soluble IL-18 receptor 1 has been reported as a biomarker in the diagnosis of rheumatoid arthritis [
31]. Its ligand, IL-18, has been associated with hepatic steatosis and elevated liver enzymes in people with HIV infection [
32] and has been used as a predictive marker in liver steatosis in obese children [
33]. Although the expression of IL-18 did not show significant difference among the three groups in our cohort, the elevated IL-18R1 may promote steatosis development through increased interaction with IL-18 in patients with CAP > 299 dB/m. SCF has been reported to regulate hemopoietic stem cells homing and proliferation [
34]. Besides that, SCF decreased in the liver injury, suggesting its proliferative effects on hepatocytes [
35]. In our study, we found significantly lower expression of SCF in patients with CAP > 299 dB/m, which might be associated with the impaired liver regeneration in these patients [
36].
Previously, our group also found that soluble inflammatory milieu was not restored even in the long-term follow up after HCV clearance upon DAA treatment with or without ribavirin [
18,
19]. This observation may explain some level of persistence of metabolic comorbidities despite viral clearance in our study. The levels of SCF and TWEAK negatively correlated with CAP value, while FGF-21 and IL-18R1 positively correlated with the CAP value at 96 w after HCV clearance (
Figure 3). This is in line with previously published studies that showed FGF-21 was increased in obesity and NAFLD [
37] and that decreased soluble TWEAK levels are associated with increased risk of diabetes [
38,
39] and metabolic syndrome [
40]. In our study, we also found that SCF, TWEAK, and IL-18R were also associated with steatosis before treatment, while FGF-21 was not. It suggests that FGF-21 may have a specific role in NASH, while the difference of SCF, TWEAK, and IL-18R at 96 w may be partially influenced by previously infected HCV. Furthermore, we previously observed the upregulation of IL-18 in NASH patients compared to healthy controls [
18], which may be associated with the increased expression of IL-18R1 at 96 w in our study. However, the expression of SCF was not significantly decreased in NASH patients [
18]. This is not consistent with the significant downregulation of SCF in patients with CAP > 299 dB/min our study. This may be due to different number of patients in the two cohort. The previous cohort contains only 5 healthy controls and 20 NASH patients, while our cohort included 94 patients. Thus, detailed effects of SCF on NASH, obesity, and metabolic syndrome need to be confirmed.
This study has several strengths and limitations as discussed below. Firstly, the study includes a well-characterized and homogenous cohort of patients, with only HCV-cleared patients included. Secondly, a large panel of cytokines and chemokines were analyzed with a very sensitive technique. On the other hand, few limitations should also be considered. Firstly, we only use CAP value for evaluating the steatosis status, which is not based on the golden standard, histology. In addition, BMI may have contributed to different CAP results as previously reported [
24]. Therefore, the findings in our study requires validation in future research. The second limitation is the lack of the mechanistic studies investigating the role of the four biomarkers in the progression of steatosis. Thirdly, only genotype 1b was included in this study. Therefore, future studies should take other genotypes into consideration.
In conclusion, a high CAP values remains in most of the patients despite successful viral clearance. Importantly, four biomarkers, namely SCF, TWEAK, FGF-21, and IL-18R1, may specifically be associated with CAP status after virus clearance.