In patients and chimpanzees infected with HCV, a robust antiviral response can be detected in the liver. A published study had previously investigated and characterized HCVcc infection of PHHs.26
However, to our knowledge, this study represents the first extensive characterization of the intrinsic innate immune response in adult PHHs following in vitro infection by HCVcc. We reproducibly show the production of IL-28 protein at the nanogram level in response to HCV nucleic acid PAMP and infectious HCVcc, and the produced IL-28 is biologically functional. In contrast, we have been unable to detect robust production of IFN-α
proteins from the infected PHHs. These results are unexpected given that the induction of the type III IFNs depends on similar signaling molecules as the type I IFNs.21
Several previous studies have described minimal type I IFN mRNA induction in cell culture and primate models of HCV infection.6,8,23
However, these studies did not specifically examine the up-regulation of type III interferons following acute HCV infection. Based on this study, we hypothesize that hepatocytes have an intrinsically enhanced ability to produce type III IFNs in response to viral stimuli in comparison to that seen with type I IFNs. Consequently, the type III IFN system is functionally the predominant antiviral pathway induced in hepatocytes.
We also explored changes in the transcriptome following infection with HCVcc in PHHs. When compared with cells treated with IL28B, it is apparent that in addition to stimulating ISGs, HCVcc up-regulates and down-regulates the expression levels of many more genes. The gene list presented in shows up-regulation of the type III IFNs in response to HCVcc and places them among the most stimulated genes that are not themselves strong ISGs, underscoring their importance in the earliest antiviral response. Pathway analysis of the microarray data was also informative as additional insight was gained into the effects of both HCVcc and IL28B on the transcriptome. In HCVcc-infected cells, one of the novel findings is that genes involved in chemotaxis were highly induced second only to the type III IFN response. Thus, in response to HCV, the hepatocytes trigger the IFN response to mitigate virus replication, while chemokine genes such as IP-10 are up-regulated to recruit immune cells from the surrounding liver parenchyma and circulation.
In this study, we addressed the functional role of the type III IFNs by determining if their production was responsible for the ISG induction following HCV infection as observed in HCV-infected chimpanzee and human liver biopsy specimens. We showed that the type III IFNs are a more likely candidate than the type I IFNs. Our data show that type III IFNs, produced by hepatocytes, may be the main driver of ISG induction following HCV infection. However, we cannot rule out a role that infiltrating peripheral blood mononuclear cells may play in producing IFNs in chronically HCV infected liver. Additional studies will have to be performed to determine if IFNs produced by peripheral blood mononuclear cells or other cell types may contribute to ISG induction in vivo. It is also interesting that the type III IFNs, in addition to inducing well-known ISGs, activate a rather distinct set of genes in PHHs from the type I IFNs, raising an intriguing possibility that the signaling pathways beyond receptor engagement may not be identical. Others have studied gene expression changes following treatment with type I and III IFNs in Huh7.5 cells and reported a more prolonged ISG induction by type III IFN.11
We observed similar results in PHHs. These data also support the involvement of type III rather than type I IFN in the induction of ISGs in HCV-infected liver because of the ability of type III IFNs to induce a prolonged upregulation of ISG mRNA as compared with type I IFN.
It is interesting to note that certain genes, like SOCS1, that function to inhibit JAK-STAT activation by type I IFN27
are highly and persistently induced by type III but not type I IFNs. These differences could explain why a high expression of ISGs may contribute to nonresponse to treatment with IFN-α
. To examine this hypothesis in HCV-infected PHHs, we tested the effect of blocking type III IFN on the anti-HCV action of type I IFN. We indeed observed a significant increase in the IFN-α
antiviral activity in the presence of blocking antibodies to type III IFN. This observation raises the intriguing possibility that nonresponders may be sensitized to the antiviral action of type I IFN through concomitant treatment with type III IFN blocking antibodies.
In conclusion, our data show robust hepatic type III IFN and other cytokine responses in several models of HCV infection. The antiviral action of type III IFN, however, may be insufficient to result in HCV clearance. The continued induction of type III IFN, with its distinct functional effects from that of type I IFN as discussed previously, may render hepatocytes relatively resistant to further type I IFN action, potentially explaining the paradoxical association of high ISG levels (marker of type III IFN production) and nonresponse to IFN-α treatment. A proposed model of the IFN response and treatment effects in HCV infection is shown in . In HCV-infected humans, cytokine responses to virus are likely determined by numerous factors including those of the host, like the IL28B genotype, and those encoded by the virus (eg, viral genotype). The interplay between the virus and host and the subsequent clinical manifestations seen following acute and chronic infection may ultimately be determined by the initial host response. Although not directly addressed in our studies, we believe that any functional role of the polymorphisms in the IL28B gene locus, in HCV responses, may be identified using these systems. More extensive analyses in a large collection of PHHs with well-defined IL-28 genotypes utilizing multiple HCV genotype strains for infection will need to be undertaken. Further characterization of cytokine responses to HCV using appropriate models will add crucial insight into the pathogenesis of HCV infection and possibly provide innovative targets for therapeutic development against hepatitis C.