Toll-like receptor 3 activation mediates the expression of interferon and interferon stimulated genes during hepadnaviral replication in HBV-transgenic mice lacking the HBs-antigen

Introduction: Chronic infection with the hepatitis B virus (HBV) is a major cause of liver-related morbidity and mortality world-wide. Previously, it has been shown that toll-like receptor (TLR) signaling is impaired by HBsAg leading to an attenuation of innate and adaptive immune responses, which may possibly facilitate chronicity of infection. Here, we have studied TLR signaling in a HBV transgenic mouse model lacking the HBs-antigen (1.4 tg HBV-s-mut) as well as in TLR3-/-/1.4 tg HBV-s-mut cross-breed animals.

Methods: Nanolipid-formulated siRNAs targeting the HBxAg sequence or the ligand for TLR3 (poly I:C), were injected intravenously into HBV-s-mut mice. HBV negative littermates were used as controls. Additionally, liver tissue of TLR3-/- and TLR3-/-/1.4 tg HBV-s-mut animals was analyzed. RNA from liver tissue was prepared and expression of HBV-RNA, interferon beta (IFN-β), interferon sensitive gene 15 (ISG15), interferon-induced protein with tetratricopeptide repeats 1 (IFI-T1), tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6) and IL-10 was determined by qtRT-PCR. HBV DNA in liver tissue was determined by Southern blot. ELISA and western blot were performed to detect HBeAg and HBcAg, respectively.

Results: Single injection of siRNAs against HBV led to suppression of HBV DNA after 48h which was sustained for more than 10 days. The HBeAg serum levels were reduced as well as hepatic HBcAg after treatment with HBxAg-targeting siRNA. The expression of IFN-β, IFI-T1 and ISG15 was up-regulated in HBV positive animals compared to control animals, which could be normalized by treatment with HBxAg-targeting siRNAs and was completely abrogated in TLR3-/-/1.4 tg HBV-s-mut. Injection of TLR3 ligand poly I:C significantly suppressed HBV replication in HBV-s-mut animals, 6h as well as 24h after application. Exposure to the TLR3 ligand induced the expression of ISG15, IL-10, IL-6 and TNF-α in HBV-s-mut mice as well as in HBV-negative littermates. However, the induction of these immune genes was significantly lowered in HBV-s-mut mice compared to HBV negative littermates. The hepatic expression of TLR3 receptor did not differ between tgHBV mice and HBV negative littermates.

Conclusions: In contrast to human liver from HBV patients, in HBV transgenic mouse model lacking HBsAg, viral replication could be associated with TLR3-mediated expression of IFNs and ISGs. Therefore, we hypothesize that HBsAg is a major component of HBV that attenuates TLR signaling thus leading to impairment of innate and adaptive immune responses in the liver. This hypothesis will be challenged in future experiments using transgenic mice that additionally express the HBsAg.