Intrahepatic Toll-Like Receptor 3 in Chronic HBV Infection Subjects: Asymptomatic Carriers, Active Chronic Hepatitis, Cirrhosis, and Hepatocellular Carcinoma

Abstract

Background: The entire disease spectrum of chronic HBV infection (CHB) includes asymptomatic carriers (AC), active chronic hepatitis (ACH), cirrhosis (Cir), and hepatocellular carcinoma (HCC). Previous study have demonstrated that the costimulation profiles from the livers of patients influenced immune responses and played various immunological roles in AC, ACH, Cir, and HCC. In addition, activation of TLR3 signaling in the liver may contribute to HBV clearance, although some HBV components are able to block TLR3 signaling and counteract HBV clearance through positive or negative feedback loops. Previous clinical studies have demonstrated that different TLR3 expressions are present in ACH patients, but no studies investigated the expression of TLR3 proteins in the livers of patients with AC, Cir, or HCC.

Objectives: This study investigated intrahepatic TLR3 expression throughout the entire disease spectrum of CHB patients and assessed the interrelations between TLR3 and costimulation proteins.

Patients and methods: Patients with ACH, Cir, HCC, and AC and healthy donors (HD) were recruited. TLR3 expression in the livers of patients were investigated using western blot analysis and immunohistochemistry. Correlations between TLR3 and costimulation proteins, including CD80, CD86, CD83, CD28, CTLA-4, CD40, and ICAM-1, were assessed.

Results: The TLR3 protein in the ACH group tended toward reduction although the P Value of the comparison between the ACH group and HD group was not statistically significant. The TLR3 levels in the HCC, AC, and Cir groups were higher than those in the HD and ACH groups. TLR3 was not interrelated with all costimulation proteins in the DCs and T cells in all five groups. No group presented any interrelation between TLR3 and CD40, except the AC group.

Conclusions: The AC, HCC, and Cir patients displayed increased levels of the intrahepatic TLR3 protein compared to the HD and AC patients. Both activation of TLR3/INF-β signaling and inhibition of TLR3/INF-β signaling by HBV components influenced TLR3 expression in the AC, ACH, Cir, and HCC subjects. However, TLR3 signaling did not influence the expression of costimulatory protein in the ACH, Cir, or HCC patients. TLR3/ IFN-β signaling did influence immune responses in the livers of CHB patients.

Keywords: Chronic Hepatitis B Infection; Costimulatory Molecule; Intrahepatic Immunopathophysiology; Toll-Like Receptors; Toll-Like Receptors 3.

Figure 1.. Quantitative Detection of TLR3 Proteins in the Liver

A, Relative quantity of TLR3 proteins in the livers from all five groups (n = 6, in each group). The images on x-ray membranes were scanned, and the relative quantity of protein was normalized to the protein quantity for each sample using β-actin protein. All the parameters are shown. Significance was defined as P < 0.05, and extreme significance was defined as P < 0.01. B, Representative western blot analysis of TLR3 proteins in the livers from six separate experiments. Homogenates obtained from the livers of patients from the five groups were subjected to electrophoresis. β-actin protein served as a protein loading standard. The molecular weight of TLR3 and β-actin was 117 kDa and 42 kDa, respectively. The TLR3 stains in the HCC, AC, and Cir patients were darker than those in the HDs or ACH subjects.

Figure 2.. Distribution of Intrahepatic TLR3 Proteins Revealed by Immunochemical Staining

Immunohistochemical staining was performed using a specific antibody against human the TLR3 protein. TLR3 stains in the livers of an ACH patient and HCC patient are shown. A, No TLR3 stains were found in the NPCs, hepatocytes, or necroinflammatory zone in the liver of the ACH patient at × 400 magnification. B, TLR3 stains with brown granules primarily appeared in the NPCs and hepatocytes, with some staining exhibited in the cells of the portal area, in the HCC subject. The NPCs with TLR3 stains surrounded the hepatocytes at × 400 magnification.