Tumor necrosis factor-α-induced protein 8-like 2 mRNA in peripheral blood mononuclear cells is associated with the disease progression of chronic hepatitis B virus infection

Hepatitis B virus (HBV) infection is a global health problem, and approximately 2 billion people are infected with HBV, of whom more than 350 million are chronically infected [1]. The natural course of chronic HBV infection often ranges from chronic hepatitis B (CHB) to liver cirrhosis (LC) and eventually to hepatocellular carcinoma (HCC) [2]. Persistent HBV infection and liver cirrhosis have been identified as leading risk factors for the development of HCC [3‐5]. It is well demonstrated that the interaction of host and HBV, especially the strength of the immune response post HBV infection, contributes to the progression of HBV infection [6].

TIPE2, tumor necrosis factor-alpha-induced protein 8 (TNFAIP8)-like 2 (TNFAIP8L2), is a newly identified negative regulator of innate immunity and cellular immunity [7]. TIPE2 can maintain immune homeostasis via negative regulation of signaling by binding to T cell receptors (TCRs) and Toll-like receptors (TLRs), and knockout of the tipe2 gene in mice leads to multiorgan inflammation, splenomegaly and premature death [7]. The high-resolution crystal structure reveals that TIPE2 contains a centrally located, large, hydrophobic central cavity, which appears to be a death effector domain (DED)-like structure, and the topology differs from caspase-8 [8]. Similar to other TNFAIP8 proteins, TIPE2 interacts with caspase-8 through their respective DED domains and promotes factor-associated suicide (Fas)-induced apoptosis [7]. In addition, TIPE2 has also been reported to control innate immunity to bacteria and double-stranded RNA (dsRNA) viruses [9], and downregulation of TIPE2 is associated with increased phagocytosis and bacterial killing [10]. Furthermore, it has been shown that TIPE2 binds to the rat sarcoma (Ras)-interacting domain to inhibit Ras-induced tumorigenesis [11]. These results suggest that TIPE2 may not only be involved in inflammation but also in cancer development.

Accelerating evidence supports the hypothesis that aberrant TIPE2 expression might play an important role in the development and progression of chronic inflammatory diseases, autoimmune disorders, stroke, diabetic nephropathy, carcinoma and atherosclerosis [12‐21]. Recent studies have revealed the downregulation of TIPE2 expression in HCC tissue compared with the paired adjacent nontumorous tissue [21]. However, the expression profiling of TIPE2 mRNA in peripheral blood mononuclear cells (PBMCs) of patients at different stages of chronic HBV infection has not yet been reported. Therefore, we aimed to investigate the dynamic expression of TIPE2 mRNA during the progression of chronic HBV infection in the present study.

Chronic HBV infections can cause immune-mediated liver damage progressing to cirrhosis and hepatocellular carcinoma (HCC) [27]. Chronic persistent inflammation in the liver may play a critical role in carcinogenesis [28‐30]. TIPE2 is a newly identified negative regulator in maintaining immune homeostasis. However, the potential roles of TIPE2 mRNA at different stages of HBV-associated liver diseases have not yet been reported. In this study, we determined the dynamic expression of TIPE2 mRNA in PBMCs in different stages of HBV-associated liver diseases and identified the potential diagnostic value of TIPE2 mRNA as a biomarker for discriminating HBV-associated HCC from CHB and LC patients.

We first reported the dynamic expression of TIPE2 mRNA during the natural history of chronic HBV infection, ranging from CHB and LC to HCC. In the present study, we demonstrated that the median levels of TIPE2 mRNA in chronic hepatitis B were significantly higher compared with those in healthy controls. These results might disagree with the previous report by Xi W et al. in 2011 in which the authors reported that TIPE2 mRNA of CHB patients was significantly downregulated compared with that of normal controls [12]. In their study, the authors defined chronic hepatitis B as HBsAg for more than 6 months but not considered liver inflammation and the immune response. Therefore, our previous study in 2017 aimed to determine the mRNA and protein levels of TIPE2 in peripheral blood and liver from CHB patients [31]. Interestingly, we reported that TIPE2 in immune clearance phases was higher than that in the immune tolerance phase, whereas TIPE2 in HBeAg-negative hepatitis was higher than that in the low replication phase, indicating that TIPE2 might contribute to the immune clearance of CHB patients [31]. In the present study, CHB patients were selected as HBsAg for more than 6 months and had elevated ALT levels, indicating that CHB patients are normally in the immune stages of immune clearance and have HBeAg-negative hepatitis. Therefore, this issue might be the reason why we obtained a discrepancy in TIPE2 expression in different populations of CHB patients [12, 31]. In addition, the healthy controls in the present study were relatively small (n = 14 for our study; n = 21 for Xi’s study [12]) for comparison, although the baseline characteristics of CHB patients and healthy controls were well matched with respect to age and sex. A larger and multicenter study with well-matched controls will be helpful in the future.

Liver cirrhosis is defined as the histological development of regenerative nodules surrounded by fibrous bands in response to chronic liver injury, which leads to portal hypertension and end-stage liver disease [32]. This is the first report to investigate the expression of TIPE2 in HBV-associated liver cirrhosis. We demonstrated that the relative level of TIPE2 mRNA in liver cirrhosis was similar to that in CHB patients, whereas the TIPE2 mRNA level in decompensated LC patients was higher than that in compensated LC patients. Furthermore, there was a significant difference in TIPE2 mRNA between LC patients with ascites and without ascites. These results indicated that TIPE2 might contribute to the progression of LC patients from the compensated phase into the decompensated phase. The MELD score is the current widely popular tool for predicting short-term mortality of liver cirrhosis [33]. We reported that TIPE2 mRNA is positively correlated with the MELD score in liver cirrhosis. This result also supports the hypothesis that TIPE2 might play an important role in the severity of liver cirrhosis. In other nonviral liver cirrhosis, TIPE2 might also exert its unique role. For example, decreased expression of TIPE2 has been reported to be associated with the hyperreactivity of monocytes to toll-like receptor ligands in primary biliary cirrhosis [34]. Functional analysis showed that TIPE2 might have protective effects on liver fibrosis by reversing the activated hepatic stellate cells [35]. Therefore, these results strongly suggest that TIPE2 mRNA is associated with liver cirrhosis. However, the exact mechanism of TIPE2 in liver cirrhosis should be well investigated in future studies.

HCC is one of the most common and rapidly fatal human malignancies in the world [36]. In the present study, we reported that the downregulation of TIPE2 mRNA expression was significantly associated with venous invasion, primary tumor size and TNM stage. In recent years, increasing studies have demonstrated that TIPE2 plays an inhibitory role in human cancer development [6, 37, 38]. A recent study showed that TIPE2 can suppress the growth and aggressiveness of hepatocellular carcinoma cells through downregulation of the phosphoinositide 3-kinase/AKT signaling pathway [39]. Another report showed that TIPE2 is an endogenous inhibitor of Rac1 in HCC, which results in attenuation of invasion and metastasis of HCC [21]. In addition, Erk1/2 and NF-κB activation has also been demonstrated to be involved in HCC progression [40]. These data suggest that TIPE2 might serve as a new target for HCC therapy.

In addition, ROC analysis has been performed to evaluate whether TIPE2 mRNA could serve as a noninvasive diagnostic biomarker. Our data showed that TIPE2 mRNA is superior to AFP for diagnosing HBV-associated HCC from CHB and LC patients, while the combination of TIPE2 mRNA and AFP conferred no advantage over TIPE2 mRNA alone for detecting HBV-associated HCC from CHB and LC patients. These results suggested that TIPE2 mRNA could serve as a useful noninvasive biomarker to diagnose HBV-associated HCC from CHB and LC patients. However, there are no data on the diagnosis of TIPE2 in the discrimination of HCC from LC and CHB patients in another cohort. Therefore, the diagnostic value of TIPE2 should be validated in more cohorts from other countries and other races in future studies. In fact, our present study is a type of cross-sectional study with the results showing that TIPE2 expression is higher in CHB and CHB with cirrhosis but lower in CHB with HCC. It seems reasonable to speculate that CHB patients with lower TIPE2 expression might be at high risk for the incidence of HCC. However, a further prospective consecutive cohort of CHB patients in a large population is essential for identifying the epidemiological effects of TIPE2 on the occurrence of HCC.

In summary, TIPE2 mRNA was associated with the disease progression of chronic HBV infection. TIPE2 mRNA might serve as a potential noninvasive biomarker to discriminate HBV-associated HCC from CHB and LC patients. However, the diagnostic value of TIPE2 should be validated and investigated in more cohorts from other countries and other races in future studies.