Toll‐like receptors, long non‐coding RNA NEAT1, and RIG‐I expression are associated with HBeAg‐positive chronic hepatitis B patients in the active phase

Background Innate immunity plays a crucial role in host‐virus interactions and greatly influences viral replication including HBV infection. However, few studies have investigated the possible antiviral immune roles played by TLRs, RIG‐I, and long no‐coding RNA NEAT1 in chronic HBV infection (CHB) patients in clinical samples and their relationships among immune responses. In this study, we sought to investigate the mRNA expression levels of TLR1‐10, RIG‐I, and NEAT1 expression in HBeAg‐positive CHB treatment‐naïve patients with the active phase. Methods The expression levels of TLR1‐10, RIG‐I, and NEAT1 of CHB patients with the active phase and healthy controls were measured by qPCR. Serum HBV DNA and routine liver biochemistry including ALT, etc were also measured to evaluate the impaired physiological function of the liver affected by CHB. Results The expression levels of TLR1 and TLR6 in CHB with active phase were remarkably lower than that in healthy controls. The levels of TLR3 in CHB patients with active phase were remarkably higher than that in healthy controls. The total NEAT1 expression was abnormally decreased in CHB patients as compared with healthy controls. The levels of RIG‐I were significantly decreased in CHB patients in the active phase when compared to healthy controls. The expression of TLR6 and RIG‐I was closely correlated with NEAT1 expression. TLR6 level was positively correlated with RIG‐I level. Conclusion Chronic HBV infection can alter the innate immune response by downregulating functional expression of TLR1, TLR6, NEAT1.


Hepatitis B virus (HBV) infection is still one of the biggest public
health challenges and over 650 000 people die each year due to HBV-associated liver diseases, such as cirrhosis and hepatocellular carcinoma. 1 Numerous evidences have shown that virological itself, host genetic background as well as immunological factors are associated with persistence of HBV infection. 2 Among the host immunological factors, innate immunity plays a crucial role in host-virus interactions, and greatly influences viral replication and the clinical outcome of HBV infection. 3 Toll-like receptors (TLRs) belong to a family of proteins named pattern recognition receptors (PRRs) and also participate in innate immunity and adaptive immunity response by recognizing invading pathogens. 4 TLRs can recognize patterns of molecules of the antigens collectively termed as pathogen-associated molecular patterns (PAMPs). 4 Previous studies demonstrated that TLR ligands may suppress HBV replication in vitro and in vivo. 5,6 Activation of TLR-mediated immunity may serve as an additional immunotherapeutic option for treating chronic HBV infection in combination with antiviral treatment. 6 HBV may interact with the TLR signaling cascade and the change in TLRs expression might be critically important for the possible explanation for escape mechanisms of virus-induced immune modulation. 3 Besides, one of the key PRRs, retinoic acid-inducible gene I (RIG-I), has attracted much attention as its role in recognizing viral dsRNA to activate the antiviral innate responses. The process of dsRNA identification RIG-I activates the downstream signaling pathways by affecting central adaptor protein (MAVS; also known as IPS-1, VISA, or Cardif) and these, in turn, lead to activation of the interferon regulatory transcription factor 3 (IRF3) and nuclear factor kappa B (NF-kB), the two important known proinflammatory transcription factors and production of type I and type III IFNs and inflammatory cytokines to cause defected immune responses against HBV. 7 In addition to the importance of TLRs and RIG-I in the immune response, recently increasing evidence has also confirmed the crucial roles of long non-coding RNAs (lncRNAs) profile in modulating host innate immunity during viral infection as well. 8,9 One of the lncRNAs, nuclear-enriched abundant transcript 1(NEAT1), was recently known as a virus-induced lncRNA (VINC) and was considered to be critical for the immune response against virus, however different virus infection, the different expression of NEAT1 observed. [8][9][10] LncRNA NEAT1 expression is upregulated after Hantaan virus (HTNV), HIV-1 infection, and HSV-1 infection, whereas it is downregulated upon Zika virus infection. [8][9][10][11] But the roles of NEAT1 in HBV infection, especially the innate immunity of chronic HBV infection at the active phase, are currently unclear.
Considering the possible antiviral immune roles played by TLRs, RIG-I, and NEAT1 in CHB patients and their interaction modulation of immune responses, it may be hypothesized that any alteration in expression of these molecules is capable to affect pathogenesis of CHB.
Consequently, here, the aim of present study was to analyze the basal mRNA expression levels of TLR1-10, RIG-I, and NEAT1 expression in HBeAg-positive CHB treatment-naïve patients with active phase subjects.    Table 1.

| Statistical analysis
The statistical analysis was performed using statistical analysis software SPSS version 16.0 (SPSS Inc, USA) and GraphPad Prism software version 5.0 (GraphPad Software, USA). Continuous variables are expressed as means ± standard deviations. Continuous variables were conducted using the Student's t test. Nonparametric statistical analysis was performed using the Mann-Whitney U test for unpaired observations. The categorical variables were analyzed using the χ 2 test. Correlations were determined using Spearman's correlation test. P < 0.05 was considered as statistical significance.

| The characteristics of CHB patients at the active phase and healthy controls
This study consisted of 40 chronic HBV patients at the active phase and 26 healthy control subjects. The clinical background of the patients and healthy control subjects were described in Table 2. As shown in Table 2, all the patients in CHB group were HBsAg and HBV DNA positive.
Comparison with control group, the serum level of total protein (TP) and albumin (Alb) in HBV group was obviously decreased. As the total protein and albumin is mainly synthesized in liver, the decreased level of these two proteins suggesting the impaired physiological function of the liver. Furthermore, liver injury index was detected. The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin (TB) were significantly increased in chronic HBV patient group compared to the healthy control group, indicating the hepatocytes injury caused by CHB infection at the active phase.

| Quantitative analysis of TLR 1-10 mRNA expression in peripheral blood
The expression levels of TLR1-10 mRNA were measured from 40 chronic HBV-infected patients and 26 healthy donors by quantitative real-time PCR (Figure 1) TLR2, TLR4, TLR5, TLR7, TLR8, TLR9, and   TLR10 shown no significant differences between the two groups.

| Downregulation of NEAT1 gene expression in peripheral blood in chronic HBV infection and healthy donors
As shown in Figure 2, the total NEAT1 expression was abnormally decreased in CHB patients as compared with healthy donors (0.4406 ± 0.02864 vs 0.6763 ± 0.07744, t = 3.291, P = 0.0016).

| A positive correlation between NEAT1, TLR, and RIG-I genes in CHB patients
To understand the possible role and status of innate immunity function during chronic HBV infection at the active phase, we further analyzed the correlation between NEAT1, TLR, and RIG-I genes.
Statistic analysis showed that the expression of TLR6 and RIG-I was closely correlated with NEAT1 mRNA expression (r = 0.4435, P = 0.0042; r = 0.4860, P = 0.0015; Figure 4A,B). It is interesting to observe that TLR6 expression levels were positively correlated with RIG-I levels (r = 0.4110, P = 0.0084, Figure 4C). It should be noted that there are several limitations in this study.

| D ISCUSS I ON
First, just the active CHB patients and health donors were included in the present study. Patients with inactive CHB patients, active CHB patients, and health donors should all be considered in further study to make the conclusion more convincing. Second, the sample size was relatively small. Therefore, more samples are needed to determine the issue discussed above in future.
In conclusion, this study suggests that chronic HBV infection can alter the innate immune response by downregulation of functional expression of TLR1, TLR6, NEAT1, and RIG-I, which may contribute to the establishment of chronic infections. Moreover, the study provides the first experimental evidence, demonstrating that the expressions of toll-like receptors, long non-coding RNA NEAT1 and RIG-I are associated with chronic HBV infection in the active phase.

ACK N OWLED G M ENTS
The study was supported by grants from National Natural Science Foundation of China (81601834, 81572067, 81702073). In addition, Yongbin Zeng wants to thank, in particular, the patience, care, support, and love from his wife, Huanhuan Huang, over the past years.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflicts of interest regarding the publication of this article.
Employment or leadership: None declared.