Human non-parenchymal liver cells produce type I and type III interferons in response to poly I:C, thereby mediating an antiviral state against HCV

Introduction: Non-parenchymal liver cells (NPC) play a crucial role in innate immunity and are involved in induction of immune tolerance. Their role in the defense against hepatotrophic viruses such as hepatitis C virus (HCV) is not well understood. Previously, this question has been mostly addressed in murine hepatocytes and NPC. Therefore, aim of the study was to characterize the Toll-like receptor (TLR)-mediated antiviral capacity in primary human Kupffer cells (KC), liver sinusoidal endothelial cells (LSEC) and hepatic stellate cells (HSC).

Methods: NPC were isolated after collagenase perfusion of liver tissue obtained from liver resections or explanted livers from HCV-infected patients or uninfected controls. Cells were isolated by density centrifugation and MACS bead separation. Cells were stimulated with TLR1 – 9 ligands for 24h, supernatants were collected and secretion of inflammatory cytokines was determined by ELISA. NPC from HCV-positive donors and healthy controls were stimulated with poly I:C for 6h, RNA was extracted and quantitative RT-PCR was performed. HCV-harboring con1 cells were co-cultured with supernatants from TLR1 – 9-activated NPC for 24h. Supernatants of poly I:C stimulated NPC were pre-incubated with neutralizing antibodies against type I, II and III interferons (IFNs), followed by co-culture with the HCV-replicon system.

Results: KC, LSEC and HSC (n = 4) secreted inflammatory cytokines IL-6, TNF-α and IL-10 in response to TLR1 – 9 agonists in a cell-type specific manner. However, only supernatants of TLR3-activated KC, LSEC and HSC mediated an antiviral activity against HCV, when co-cultured with the HCV replicon system con1. Treatment of NPC with TLR3 agonist poly I:C led to a significant induction of IFN-β, IL-28A/IL-28B and IL-29 in KC, LSEC and HSC. Furthermore, LSEC but not KC or HSC isolated from HCV-infected donors revealed higher responsiveness to poly I:C, represented by higher expression levels of IFN-β, IL-28A, IL-28B, IL-29 compared to those of uninfected controls. The antiviral effect of supernatants from TLR3-activated NPC was not impeded by pre-incubation with neutralizing antibodies against type I, II or III IFNs.

Conclusions: NPC respond to TLR ligands by production of inflammatory cytokines. A TLR-induced antiviral effect in these cells, however, is restricted to TLR3 and seems to be mediated by the combination of type-I and type-III IFNs. In accordance with data recently obtained from primary human hepatocytes, TLR3-mediated expression of IFN-β, IL-28A, IL-28B and IL-29 is also elevated in LSEC obtained from HCV-infected patients, compared to uninfected controls. These findings shed new light on the relevance of NPC in the pathogenesis of HCV.