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Persistent hepatitis B viral (HBV) infection results in chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC). An efficient control of virus infections requires the coordinated actions of both innate and adaptive immune responses. In order to define the role of innate immunity effectors against HBV, viral clearance was studied in a panel of immunodeficient mouse strains by the hydrodynamic injection approach. Our results demonstrate that HBV viral clearance is not changed in IFN-α/β receptor (IFNAR), RIG-I, MDA5, MYD88, NLRP3, ASC, and IL-1R knock-out mice, indicating that these innate immunity effectors are not required for HBV clearance. In contrast, HBV persists in the absence of tumor necrosis factor-alpha (TNF-α) or in mice treated with the soluble TNF receptor blocker, Etanercept. In these mice, there was an increase in PD-1-expressing CD8+ T-cells and an increase of serum HBV DNA, HBV core, and surface antigen expression as well as viral replication within the liver. Furthermore, the induction of TNF-α in clearing HBV is dependent on the HBV core, and TNF blockage eliminated HBV core-induced viral clearance effects. Finally, the intra-hepatic leukocytes (IHLs), but not the hepatocytes, are the cell source responsible for TNF-α production induced by HBcAg. These results provide evidences for TNF-α mediated innate immune mechanisms in HBV clearance and explain the mechanism of HBV reactivation during therapy with TNF blockage agents.

Human osteoclast formation from mononuclear phagocyte precursors involves interactions between tumor necrosis factor (TNF) ligand superfamily members and their receptors. Recent evidence indicates that in addition to triggering apoptosis, the TNF-related apoptosis-inducing ligand (TRAIL) induces osteoclast differentiation. To understand TRAIL-mediated signal transduction mechanism in osteoclastogenesis, we demonstrated that TRAIL induces osteoclast differentiation via a Tumor necrosis factor receptor-associated factor 6 (TRAF-6)-dependent signaling pathway. TRAIL-induced osteoclast differentiation was significantly inhibited by treatment with TRAF-6 siRNA and TRAF6 decoy peptides in both human monocytes and murine RAW264.7 macrophage cell lines, as evaluated in terms of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and bone resorption activity. Moreover, TRAIL-induced osteoclast differentiation was also abolished in TRAF6 knockout bone marrow macrophages. In addition to induction of NFATc1, treatment of TRAIL also induced ubiquitination of TRAF6 in osteoclast differentiation. Thus, our data demonstrate that TRAIL induces osteoclastic differentiation via a TRAF-6 dependent signaling pathway. This study suggests TRAF6-dependent signaling may be a central pathway in osteoclast differentiation, and that TNF superfamily molecules other than RANKL may modify RANK signaling by interaction with TRAF6-associated signaling.

Persistent hepatitis B viral (HBV) infection results in chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC). Recent studies in animal models of viral infection indicate that the interaction between the inhibitory receptor, programmed death (PD)-1, on lymphocytes and its ligand (PD-L1) play a critical role in T-cell exhaustion by inducing T-cell inactivation. High PD-1 expression levels by peripheral T-lymphocytes and the possibility of improving T-cell function by blocking PD-1-mediated signaling confirm the importance of this inhibitory pathway in inducing T-cell exhaustion. We studied T-cell exhaustion and the effects of PD-1 and PD-L1 blockade on intrahepatic infiltrating T-cells in our recently developed mouse model of HBV persistence. In this mouse animal model, we demonstrated that there were increased intrahepatic PD-1-expressing CD8+ and CD4+ T cells in mice with HBV persistence, but PD-1 upregulation was resolved in mice which had cleared HBV. The Intrahepatic CD8+ T-cells expressed higher levels of PD-1 and lower levels of CD127 in mice with HBV persistence. Blockade of PD-1/PD-L1 interactions increased HBcAg-specific interferon (IFN)-γ production in intrahepatic T lymphocytes. Furthermore, blocking the interaction of PD-1 with PD-L1 by an anti-PD-1 monoclonal antibody (mAb) reversed the exhausted phenotype in intrahepatic T lymphocytes and viral persistence to clearance of HBV in vivo. Our results indicated that PD-1 blockage reverses immune dysfunction and viral persistence of HBV infection in a mouse animal model, suggesting that the anti-PD-1 mAb might be a good therapeutic candidate for chronic HBV infection.