The present study demonstrates the role of type I IFN in the induction of functional immunoproteasomes in the target organ of the CD8 T cell response during a viral infection. Not only exogenous IFN-α and IFN-β, but also endogenous production of type I IFN by intracellular HCV RNA or poly(I:C), stimulated induction of functionally active immunoproteasomes in vitro (Figures –). Prospective analysis of liver biopsies from HCV-infected chimpanzees demonstrated that this mechanism was also operative in vivo during the early weeks of HCV infection (Figure ). Collectively, these results challenge the current dogma of IFN-γ being the sole inducer of immunoproteasomes during a viral infection.
Our findings may represent a more general antiviral response, because dsRNA-induced, RIG-I–mediated type I IFN responses in virus-infected cells have also been described for other RNA viruses such as Newcastle disease virus (21
), vesicular stomatitis virus (22
), and paramyxovirus (23
). Type I IFN–mediated induction of immunoproteasomes was especially evident in HCV infection, where intrahepatic expression of IFN-γ was detectable very late, i.e., not earlier than 5–8 weeks after infection (Figure ). Thus, the innate type I IFN response not only exerts antiviral functions but also prepares the infected target organ for the adaptive immune response, in particular by immunoproteasome-dependent antigen processing. This mechanism complements a similar role of the innate immune response by type I IFN–induced upregulation of MHC molecules that has been described previously (24
). Both mechanisms may increase recognition of infected cells by incoming CD8 T cells and elevate virus-infected cells from passive targets to active facilitators of the adaptive immune response.
The type I IFN–mediated induction of immunoproteasomes described herein differed from the results of a previous publication that described normal induction of immunoproteasomes in LCMV-infected mice that lack the IFN-α/βR (11
). In addition to virus and host differences between both models, several additional factors may explain why a type I IFN effect was not observed in the LCMV model. First, the effect of type I IFN may have been masked by an early and vigorous IFN-γ response in the LCMV model. The temporal dissociation between early type I IFN response and late IFN-γ response in the HCV model was essential to recognize the additional and specific effect of type I IFN. Second, human hepatocytes differ from murine hepatocytes in regard to IFN-α/βR expression and responsiveness to type I IFN (26
). Whereas human hepatocytes express functional IFN-α/βR 2c, murine hepatocytes predominantly express IFN-α/βR 2a, which exerts inhibitory functions and blunts the effect of type I IFN on murine hepatocytes (26
). Third, the new class of IFN-λ1 (IL-29), IFN-λ2 (IL-28A), and IFN-λ3 (IL-28B) (27
), which activates the same JAK/STAT signaling pathway as type I IFN, may have induced immunoproteasomes in IFN-α/βR–/–
mice during LCMV infection.
Our findings are supported by microarray studies of gene expression patterns in acute hepatitis C, which also detect early expression of immunoproteasome subunits (29
). In addition, they are indirectly supported by data from acute HBV infection (31
). In contrast to acute HCV infection, acute HBV infection does not induce any detectable type I IFN response in the liver, as determined by lack of 2,5-OAS-1 mRNA induction in serial liver biopsies of HBV-infected chimpanzees (31
). Consistent with the absence of a type I IFN response, no increased expression of immunoproteasome subunits was observed during the early phase of acute HBV infection (31
). In fact, immunoproteasome subunits were only induced in the later phase of HBV infection, concomitant with an increase in intrahepatic IFN-γ mRNA levels (31
What role does the induction of immunoproteasomes by type I IFN play in the course of acute HCV infection? Both the kinetics and the peak levels of intrahepatic immunoproteasome subunit mRNA expression were comparable in all 5 chimpanzees in our study (Figure ). Likewise, kinetics and peak levels of intrahepatic 2,5-OAS-1 and type I IFN mRNA levels were comparable in all 5 chimpanzees (Figure ). These findings suggest that differences in the outcome of HCV infection do not solely depend on the presence or absence of type I IFN–mediated induction of immunoproteasomes. However, the timing of type I IFN–mediated induction of immunoproteasomes in relation to the adaptive CD8 T cell response in the liver may be important. An effective CD8 T cell response is an important factor for the outcome of HCV infection, as previously shown by us (32
) and others (15
). An increasing gap between transient early type I IFN–mediated induction of immunoproteasomes and late CD8 T cell infiltration in the liver may render the antigen recognition by intrahepatic HCV-specific T cells suboptimal. Interestingly, the timing of the intrahepatic increase of IFN-γ mRNA levels as indicated by the dashed vertical lines in Figure (which correlates closely with intrahepatic CD8β mRNA levels; Figure ), occurs later in chimpanzees with a chronically evolving course of HCV infection. This is consistent with a previously described delay in ALT peak, another correlate of CD8 T cell activity in the liver, in a larger group of chimpanzees with a chronic outcome of infection (44
This timely dissociation between early transient immunoproteasome expression in the liver and late infiltration of CD8 T cells may be overcome by repeated exogenous administration of high doses of type I IFN, as impressively shown by the high (95%) HCV clearance rates if recombinant IFN-α is administered during the first 6 months of HCV infection (45
). The high treatment response rate cannot readily be explained by IFN’s antiviral effects, because clearance rates are much lower in chronic HCV infection (46
), when immune escape mechanisms are already established (16
) and the HCV-specific T cell response is exhausted (15
Thus, it is possible that IFN-α–mediated induction of immunoproteasomes and better antigen recognition by liver-infiltrating T cells contribute to the high effectiveness of type I IFN–based therapies (95%) if administered early during HCV infection. Practically, however, it is very difficult to prove this hypothesis in a clinical setting, because it would require multiple liver biopsies per patient during the acute phase of hepatitis C. Chimpanzees are not an appropriate model for this purpose because their HCV titers do not decrease during therapy with recombinant human IFN-α (50
We conclude that intrahepatic induction of immunoproteasomes precedes the intrahepatic expression of IFN-γ in this clinically relevant model of viral infection in humans. Type I IFN secreted in response to dsRNA in virus-infected hepatocytes induced functional immunoproteasomes with characteristic proteolytic activity.